Method for blocking binding of CX3C chemokines using CX3C chemokine antibodies

ABSTRACT

Nucleic acids encoding a new family of chemokines, the CX3C family, from a mammal, reagents related thereto, including specific antibodies, and purified proteins are described. Methods of using said reagents and related diagnostic kits are also provided.

The present application is a divisional of U.S. patent application Ser.No. 12/833,069, filed Jul. 9, 2010; which is a continuation of U.S.patent application Ser. No. 12/510,586, filed Jul. 28, 2009; which is acontinuation of U.S. patent application Ser. No. 12/198,626, filed Aug.26, 2008; which is a continuation of U.S. patent application Ser. No.11/495,801, filed Jul. 28, 2006; which is a continuation of U.S. patentapplication Ser. No. 10/397,559, filed Mar. 25, 2003; which is adivision of U.S. patent application Ser. No. 09/771,023, filed Jan. 25,2001, now U.S. Pat. No. 6,566,503; which is a division of U.S. patentapplication Ser. No. 09/093,482, filed Jun. 8, 1998; which is a divisionof U.S. patent application Ser. No. 08/786,068, filed Jan. 21, 1997;which is a continuation of U.S. patent application Ser. No. 08/649,006,filed May 16, 1996, now U.S. Pat. No. 6,548,654; which is a continuationof U.S. patent application Ser. No. 08/590,828 filed Jan. 24, 1996, eachof which is incorporated herein by reference in its entirety.

FIELD OF THE INVENTION

The present invention contemplates compositions related to proteinswhich function in controlling development, differentiation, trafficking,and physiology of mammalian cells, e.g., cells of a mammalian immunesystem. In particular, it provides proteins which regulate or evidencedevelopment, differentiation, and function of various cell types,including hematopoietic cells.

BACKGROUND OF THE INVENTION

The circulating component of the mammalian circulatory system comprisesvarious cell types, including red and white blood cells of the erythroidand myeloid cell lineages. See, e.g., Rapaport (1987) Introduction toHematology (2d ed.) Lippincott, Philadelphia, Pa.; Jandl (1987) Blood:Textbook of Hematology, Little, Brown and Co., Boston, Mass.; and Paul(ed.) (1993) Fundamental Immunology (3d ed.) Raven Press, N.Y.

For some time, it has been known that the mammalian immune response isbased on a series of complex cellular interactions, called the “immunenetwork.” Recent research has provided new insights into the innerworkings of this network. While it remains clear that much of theresponse does, in fact, revolve around the network-like interactions oflymphocytes, macrophages, granulocytes, and other cells, immunologistsnow generally hold the opinion that soluble proteins, known aslymphokines, cytokines, or monokines, play a critical role incontrolling these cellular interactions. Thus, there is considerableinterest in the isolation, characterization, and mechanisms of action ofcell modulatory factors, an understanding of which should lead tosignificant advancements in the diagnosis and therapy of numerousmedical abnormalities, e.g., immune system and other disorders.

Lymphokines apparently mediate cellular activities in a variety of ways.They have been shown to support the proliferation, growth, anddifferentiation of pluripotential hematopoietic stem cells into vastnumbers of progenitors comprising diverse cellular lineages making up acomplex immune system. These interactions between cellular componentsare necessary for a healthy immune response. These different cellularlineages often respond in a different manner when lymphokines areadministered in conjunction with other agents.

The chemokines are a large and diverse superfamily of proteins. Thesuperfamily is subdivided into three branches, based upon whether thefirst two cysteines in the classical chemokine motif are adjacent(termed the “C—C” branch) or spaced by an intervening residue (“C—X—C”),or a new branch which lacks two cysteines in the corresponding motif,represented by the chemokines known as lymphotactins. See, e.g., Schalland Bacon (1994) Current Opinion in Immunology 6:865-873; and Bacon andSchall (1996) Int. Arch. Allergy & Immunol. 109:97-109.

Many factors have been identified which influence the differentiationprocess of precursor cells, or regulate the physiology or migrationproperties of specific cell types. These observations indicate thatother factors exist whose functions in immune function were heretoforeunrecognized. These factors provide for biological activities whosespectra of effects may be distinct from known differentiation oractivation factors. The absence of knowledge about the structural,biological, and physiological properties of the regulatory factors whichregulate cell physiology in vivo prevents the modification of theeffects of such factors. Thus, medical conditions where regulation ofthe development or physiology of relevant cells is inappropriate remainunmanageable.

SUMMARY OF THE INVENTION

The present invention reveals the existence of a previously unknownclass of chemokine-motif containing molecules which are herebydesignated the CX3C chemokines. The CX3Ckines have three amino acidswhich separate the cysteines in the corresponding region of thechemokine motif. Based on sequence analysis of the two CX3C proteinsequences described below, it is apparent that the CX3Ckines do notbelong to the C, C—C, or C—X—C chemokine families. They represent thefirst known members of a new heretofore unidentified class of chemokinesdesignated CX3Ckines, or alternatively, the CX3C family of chemokines.

The present invention provides a composition of matter selected from anantibody binding site which specifically binds to a mammalian CX3Cchemokine; an expression vector encoding a mammalian CX3C chemokine orfragment thereof; a substantially pure protein which is specificallyrecognized by the antibody binding site; and a substantially pure CX3Cchemokine or peptide thereof, or a fusion protein comprising a 30 aminoacid fragment of CX3C chemokine sequence.

In the antibody binding site embodiments, the antibody binding site maybe: specifically immunoreactive with a mature protein selected from thegroup consisting of the polypeptides of SEQ ID NO: 2, 4, 6 and 8; raisedagainst a purified or recombinantly produced human or mouse CX3Cchemokine; in a monoclonal antibody, Fab, or F(ab)₂; immunoreactive withdenatured antigen; or in a labeled antibody. In certain embodiments; theantibody binding site is detected in a biological sample by a method of:contacting a binding agent having an affinity for CX3C chemokine proteinwith the biological sample; incubating the binding agent with thebiological sample to form a binding agent:CX3C chemokine proteincomplex; and detecting the complex. In a preferred embodiment, thebiological sample is human, and the binding agent is an antibody.

A kit embodiment is provided possessing a composition, described above,with either instructional material for the use of the composition; orsegregation of the composition into a container.

A nucleic acid embodiment of the invention includes an expression vectorencoding a CX3C chemokine protein, wherein the protein specificallybinds an antibody generated against an immunogen selected from themature polypeptide portions of SEQ ID NO: 2, 4, 6, and 8. The vectormay: encode a CX3C chemokine polypeptide with complete sequence identityto a naturally occurring human CX3C chemokine domain; encode a CX3Cchemokine protein comprising sequence selected from the polypeptides ofSEQ ID NO: 2, 4, 6, and 8; or comprise sequence selected from thenucleic acids of SEQ ID NO: 1, 3, 5, or 7. In other embodiments, thevector is capable of selectively hybridizing to a nucleic acid encodinga CX3C chemokine protein, e.g., a mature protein coding segment of SEQID NO: 1, 3, 5, or 7. In various preferred embodiments, the isolatednucleic acid is detected in a biological sample by a method: contactinga biological sample with a nucleic acid probe capable of selectivelyhybridizing to the nucleic acid; incubating the nucleic acid probe withthe biological sample to form a hybrid of the nucleic acid probe withcomplementary nucleic acid sequences present in the biological sample;and determining the extent of hybridization of the nucleic acid probe tothe complementary nucleic acid sequences. In such method, preferably thenucleic acid probe is capable of hybridizing to a nucleic acid encodinga protein consisting of the polypeptides of SEQ ID NO: 2, 4, 6, or 8

In protein embodiments, the isolated CX3C chemokine protein willpreferably be of approximately 11,000 to 15,000 daltons when inunglycosylated form, and the CX3C chemokine protein specifically bindsto an antibody generated against an immunogen; the polypeptides of SEQID NO: 2, 4, 6, or 8; and the CX3C chemokine lacks the cysteinestructural motifs and sequence characteristic of a C, a CC, or a CXCchemokine. In various embodiments, the isolated CX3C chemokine proteinis: selected from human CX3Ckine or mouse CX3Ckine; consists of apolypeptide comprising sequence from SEQ ID NO: 2, 4, 6, or 8;recombinantly produced, or a naturally occurring protein.

The present invention also embraces a cell transfected with the nucleicacid encoding a CX3C chemokine, e.g., where the nucleic acid has SEQ IDNO: 1, 3, 5, or 7.

The invention also provides a method of modulating physiology ordevelopment of a cell by contacting the cell with a CX3C chemokine, oran antagonist of the chemokine. In preferred embodiments, the physiologyis attraction, and the cell is a peripheral blood monocyte or a T cell.

DETAILED DESCRIPTION I. General

The present invention provides DNA sequences encoding mammalian proteinswhich exhibit structural properties or motifs characteristic of acytokine or chemokine. For a review of the chemokine family, see, e.g.,Lodi, et al. (1994) Science 263:1762-1767; Gronenborn and Clore (1991)Protein Engineering 4:263-269; Miller and Kranger (1992) Proc. Nat'lAcad. Sci. USA 89:2950-2954; Matsushima and Oppenheim (1989) Cytokine1:2-13; Stoeckle and Baker (1990) New Biol. 2:313-323; Oppenheim, et al.(1991) Ann. Rev. Immunol. 9:617-648; Schall (1991) Cytokine 3:165-183;and The Cytokine Handbook Academic Press, NY. The proteins describedherein are designated CX3Ckines because they were initially recognizedas sharing significant structural features of chemokines, but whosestructural features also exhibit sequence peculiarity, e.g., structuralmotifs, distinct from the other known branches of the chemokinemolecules.

The best characterized embodiment of this family of proteins wasdiscovered from a human and is designated human CX3C chemokine (GenBankAccession No. H14940). See, SEQ ID NO: 1-4 An additional CX3Ckine,represented by a mouse molecule, designated mouse CX3Ckine, is alsodescribed herein. See Table 1 and SEQ ID NO: 5-8. The descriptions beloware directed, for exemplary purposes, to primate and rodent embodiments,e.g., human and mouse, but are likewise applicable to relatedembodiments from other, e.g., natural, sources. These sources shouldinclude various vertebrates, typically warm blooded animals, e.g., birdsand mammals, particularly domestic animals, and primates.

In the human sequence (SEQ ID NO: 1-4), the signal sequence runs fromabout Met1 to Gly24, thus the mature polypeptide begins at about Gln25and ends at about Val 397. A chemokine domain runs from about Gln25 toabout Gly100; a stalk region, which possesses many potentialglycosylation sites, runs from about Gly101 to about Gln341; atranmembrane region begins at about Ala342 and ends at about Thr361; andan intracellular domain, containing two tyrosine phosphorylation sitesat residues 382 and 392, runs from about Tyr362 to Val397.

TABLE 1 Mouse CX3C chemokine nucleic acid (SEQ ID NO: 7) andamino acid (SEQ ID NO: 8) sequences. The coding sequence runsfrom nucleotides 62-1249. The signal sequence runs from aboutMet1 through Gly24. The mature polypeptide runs from aboutGln25 through Val395. The chemokine domain runs from aboutGln25 through Gly100. The stalk region runs from about Gly101through Gln339. The transmembrane domain runs from about Ala340througn Phe358. The cytoplasmic domain runs from about Ala359through Val395.TGACTACTAG GAGCTGCGAC ACGGCCCAGC CTCCTGGCCG CCGAATTCCT GCACTCCAGC   60C ATG GCT CCC TCG CCG CTC GCG TGG CTG CTG CGC CTG GCC GCG TTC  106  Met Ala Pro Ser Pro Leu Ala Trp Leu Leu Arg Leu Ala Ala Phe    1               5                  10                  15TTC CAT TTG TGT ACT CTG CTG CCG GGT CAG CAC CTC GGC ATG ACG AAA  154Phe His Leu Cys Thr Leu Leu Pro Gly Gln His Leu Gly Met Thr Lys                 20                  25                  30TGC GAA ATC ATG TGC GGC AAG ATG ACC TCA CGA ATC CCA GTG GCT TTG  202Cys Glu Ile Met Cys Gly Lys Met Thr Ser Arg Ile Pro Val Ala Leu             35                  40                  45CTC ATC CGC TAT CAG CTA AAT CAG GAG TCC TGC GGC AAG CGT GCC ATT  250Leu Ile Arg Tyr Gln Leu Asn Gln Glu Ser Cys Gly Lys Arg Ala Ile         50                  55                  60GTC CTG GAG ACG ACA CAG CAC AGA CGC TTC TGT GCT GAC CCG AAG GAG  298Val Leu Glu Thr Thr Gln His Arg Arg Phe Cys Ala Asp Pro Lys Glu     65                  70                  75AAA TGG GTC CAA GAC GCC ATG AAG CAT CTG GAT CAC CAG GCT GCT GCC  346Lys Trp Val Gln Asp Ala Met Lys His Leu Asp His Gln Ala Ala Ala 80                  85                  90                  95CTC ACT AAA AAT GGT GGC AAG TTT GAG AAG CGG GTG GAC AAT GTG ACA  394Leu Thr Lys Asn Gly Gly Lys Phe Glu Lys Arg Val Asp Asn Val Thr                100                 105                 110CCT GGG ATC ACC TTG GCC ACT AGG GGA CTG TCC CCA TCT GCC CTG ACA  442Pro Gly Ile Thr Leu Ala Thr Arg Gly Leu Ser Pro Ser Ala Leu Thr            115                 120                 125AAG CCT GAA TCC GCC ACA TTG GAA GAC CTT GCT TTG GAA CTG ACT ACT  490Lys Pro Glu Ser Ala Thr Leu Glu Asp Leu Ala Leu Glu Leu Thr Thr        130                 135                 140ATT TCC CAG GAG GCC AGG GGG ACC ATG GGG ACT TCC CAA GAG CCA CCG  538Ile Ser Gln Glu Ala Arg Gly Thr Met Gly Thr Ser Gln Glu Pro Pro    145                 150                 155GCA GCA GTG ACC GGA TCA TCT CTC TCA ACT TCC GAG GCA CAG GAT GCA  586Ala Ala Val Thr Gly Ser Ser Leu Ser Thr Ser Glu Ala Gln Asp Ala160                 165                 170                 175GGG CTT ACG GCT AAG CCT CAG AGC ATT GGA AGT TTT GAG GCG GCT GAC  634Gly Leu Thr Ala Lys Pro Gln Ser Ile Gly Ser Phe Glu Ala Ala Asp                180                 185                 190ATC TCC ACC ACC GTT TGG CCG AGT CCT GCT GTC TAC CAA TCT GGA TCT  682Ile Ser Thr Thr Val Trp Pro Ser Pro Ala Val Tyr Gln Ser Gly Ser            195                 200                 205AGC TCC TGG GCT GAG GAA AAA GCT ACT GAG TCC CCC TCC ACT ACA GCC  730Ser Ser Trp Ala Glu Glu Lys Ala Thr Glu Ser Pro Ser Thr Thr Ala        210                 215                 220CCA TCT CCT CAG GTG TCC ACT ACT TCA CCT TCA ACC CCA GAG GAA AAT  778Pro Ser Pro Gln Val Ser Thr Thr Ser Pro Ser Thr Pro Glu Glu Asn    225                 230                 235GTT GGG TCC GAA GGC CAA CCC CCA TGG GTC CAG GGA CAG GAC CTC AGT  826Val Gly Ser Glu Gly Gln Pro Pro Trp Val Gln Gly Gln Asp Leu Ser240                 245                 250                 255CCA GAG AAG TCT CTA GGG TCT GAG GAG ATA AAC CCA GTT CAT ACT GAT  874Pro Glu Lys Ser Leu Gly Ser Glu Glu Ile Asn Pro Val His Thr Asp                260                 265                 270AAT TTC CAG GAG AGG GGG CCT GGC AAC ACA GTC CAC CCC TCA GTG GCT  922Asn Phe Gln Glu Arg Gly Pro Gly Asn Thr Val His Pro Ser Val Ala            275                 280                 285CCC ATC TCC TCT GAA GAG ACC CCC AGC CCA GAG CTG GTG GCC TCG GGC  970Pro Ile Ser Ser Glu Glu Thr Pro Ser Pro Glu Leu Val Ala Ser Gly        290                 295                 300AGC CAG GCT CCT AAG ATA GAG GAA CCC ATC CAT GCC ACT GCA GAT CCC 1018Ser Gln Ala Pro Lys Ile Glu Glu Pro Ile His Ala Thr Ala Asp Pro    305                 310                 315CAG AAA CTG AGT GTG CTT ATC ACT CCT GTC CCC GAC ACC CAG GCA GCC 1066Gln Lys Leu Ser Val Leu Ile Thr Pro Val Pro Asp Thr Gln Ala Ala320                 325                 330                 335ACA AGG AGG CAG GCA GTG GGG CTA CTG GCT TTC CTT GGT CTT CTT TTC 1114Thr Arg Arg Gln Ala Val Gly Leu Leu Ala Phe Leu Gly Leu Leu Phe                340                 345                 350TGC CTA GGG GTG GCC ATG TTT GCT TAC CAG AGC CTT CAG GGC TGT CCC 1162Cys Leu Gly Val Ala Met Phe Ala Tyr Gln Ser Leu Gln Gly Cys Pro            355                 360                 365CGC AAA ATG GCG GGG GAA ATG GTA GAA GGC CTC CGC TAC GTC CCC CGT 1210Arg Lys Met Ala Gly Glu Met Val Glu Gly Leu Arg Tyr Val Pro Arg        370                 375                 380AGC TGT GGC AGT AAC TCA TAC GTC CTG GTG CCA GTG TGA GCTGCTTGCC 1259Ser Cys Gly Ser Asn Ser Tyr Val Leu Val Pro Val *    385                 390                 395TGCCTGCCTG TGTCCAGAGT GTGATTCGGA CAGCTGTCTG GGGACCCCCC CCCATCCTCA 1319TACCCACCTT CATCCACGCT GGGGAAATGG GAATGGAGAA GCTGGACCTC CAGGGGCTGT 1379GGGCTCCATC CAATCCCCCT TCCCCCGAGG GGTGGCCCCG GAGGCCACCC TAGACCACTA 1439TTCACTTATC AGAGACAGAG CAGGTGACCT TCCAGCTCCT CTATATTTGA AAGAATCCTC 1499TGCTGCTGGC TGGTTAGAGG GGCCCTTGAC ACCCCAACTC CAGTGAACAA TTATTTATTG 1559GATTCCCAGC CCCTGCGACG ACACCTGTTT CCCGCGCGCA CCGTGGTCCG CCCATATCAC 1619AAGCAGCAGG CCAGGCCTAT CTGCCTGTCC CCCTGACCTC CTTGTGTCTC CTGGCTTTGC 1679TGCAGTCGCC AGCCCTTCTC CTCCCCGGCC AGCCGCGGTG CTATCTGCCC TATGTCTCCC 1739TCTATCCCCT GTACAGAGCG CACCACCATC ACCATCAACA CCGCTGTTGT GTCTTTTCTT 1799GCATGAGGTT AAAGCTGTGT TTTCTGGAGC TCTCCGGGAA GGGAGACAAG CTTGCGAGAG 1859GGTTTAAAGT GTTCCTCCCC AGACTTGGAT GTGCTGTGAG GGCATGCTGC GTCTGAAGGA 1919AGGGTCCAGT CCCCACTCGG CTACCAGCAC CACAAAGTGC CCCACCTGTA AAAGGAAAGA 1979AACGTGGTCC AGAGCTGGCA ATAACCTATG GCCCTGACAT CATCACTTTC TCTGAGATCC 2039TTGTCTCCAC CCCTGGGTGC AACCCCACCC CTTATCAACA TTAATAGTCA CTGCCATTCC 2099ACTGGACTGA CATTTTTGTA CCCTGTGATT CTGAGGGCTG GCAAGGAGTG GCTTGAGAGT 2159GCAGATCGTA CCCTGTATGC CCCCCCCAAA TGGAGGCTGA GTTGGGGACT TGCAGGAACA 2219GAGGCCAACT CAGATGGCTT CCCCTGTGTT CTCACTAGAA ACCCCTCCCC CATGCACCAA 2279GGTGACAGTC ACAGGTCTGC CCTGGCTAAA GGACAAGCCA CATAGGAAAG ATTAGGACAA 2339GCCCCTCGGA GGCAGAGGAT CCAGGGTAAA CCCCTGGAGT GGCCACAAAC CCAATTTCAG 2399TGTAGGGACT TGTGCATGTG TGTACTTGCA TAGTCAGACA GAGGCTGCCA GGGTCCTTTC 2459CTGTCTCTGA GAGCAGTGTT CACGCCAAGG ACTCACCTTT GCCCCCATTG CAGGCAGGGC 2519CAGAACTCCC ATAGCATTCT CCAAGAGCCC TGTGACATTT TCTGGAAGGA ACTCTGCCCT 2579GGGCGCAAAG TGACTGCTGA AGCAAGGAGC AGCTGAGCAG CACCCCAGCG GAGCTGAGCC 2639GGCAGGCCAC GCCCCTCGGG GGGGGGCATT TCTACCCGCC CTGCTCTGAA TAGCTCCAAC 2699TTCACCTTAG GAGCCTCCCA GGGGCGAGCT TCACCCAGAA GCCAGTGACT CACTCCTTGA 2759TTGGTGGAAG CTCAGTTGGC TCCTGAGAGT GAGGAAGCCA ACCCTTTGTC GACCCTCCTC 2819CTGGGAAGCC TGTGGGCGGC TCTGATCATG CTCCACAGAA CCAGTTGTAG GCCTGAGCCG 2879CAGCAGCCCG AGTGCACTAT ATCCTGGCTC CTTCGGTGGG GAACCTTTAA GGGTTGGGAC 2939ACCCGTCATC GGACTTTGTT GGTTCCTCCC TCCCAGAGCA GAATGTGGGC CGTAACAATC 2999TGAGGAGGAC TTTAAAAGTT GTTGATCCTT TAGGGTTTTT TTTCAAGCAT CATTACCAAT 3059GTCTGT 3065

The CX3Ckine proteins of this invention are defined in part by theirphysicochemical and biological properties. The biological properties ofthe human and mouse CX3Ckines described herein, e.g., human CX3Ckine andmouse CX3Ckine, are defined by their amino acid sequence, and maturesize. They also should share biological properties. The human and mouseCX3Ckine molecules exhibit about 70-80% amino acid identity, dependingon whether the signal or mature sequences are compared. One of skillwill readily recognize that some sequence variations may be tolerated,e.g., conservative substitutions or positions remote from the helicalstructures, without altering significantly the biological activity ofthe molecule.

Table 2 shows a sequence alignment of human CX3Ckine amino acid sequence(CX3C) with the C—X—C chemokine Groa (Gro), the C chemokine lymphotactin(LTn), and the C—C chemokine Macrophage inflammatory protein 1β(MIP-1β).

TABLE 2 Comparison of various chemokines Exon 1 GroMIPATRSLLCAALLLLATSRLATG (SEQ ID NO: 9) LTn      MRLLLLTFLGVCCLTPWVV(SEQ ID NO: 10) MIP-1β  MKLCVSALSLLLLVAAFCAPGFS (SEQ ID NO: 11) CX3MAPISLSWLLRLATFCHLTVLLAG (SEQ ID NO: 2) Exon 2 Gro  APIANELRCQCLQTMA.GIHLKNIQSLKVLPSGPHCTQT LTnEGVGTEVLEESSCVNLQTQRLPVQKIKTYIIWEG....AMR MIP APMGSDPPTSCCFSYTARKLPRNFVVDYYETSSL..CSQP CX3 QHHGVTKCNITC.SKMTSKIPVALLIHYQQNQAS..CGKR Exon 3 GROEVIATLKNGREACLDPEAPLVQKIVQKMLKGVPK LTNAVIFVTKRGLKICADPEAKWVLAAIKTVDGRASTRKNMAETVPGTGAQRSTSTAITLTG MIPAVVFQTKRSKQVCADPSESWVQEYVYDLELN CX3AIILETRQHRLFCADPKEQWVKDAMQHLDRQAAALTRNG . . .

CX3Ckines are present in specific tissue types, e.g., neural tissues,and the interaction of the protein with a receptor will be important formediating various aspects of cellular physiology or development. Thecellular types which express message encoding CX3Ckines suggest thatsignals important in cell differentiation and development are mediatedby them. See, e.g., Gilbert (1991) Developmental Biology (3d ed.)Sinauer Associates, Sunderland, M A; Browder, et al. (1991)Developmental Biology (3d ed.) Saunders, Philadelphia, Pa.; Russo, etal. (1992) Development: The Molecular Genetic Approach Springer-Verlag,New York, N.Y.; and Wilkins (1993) Genetic Analysis of AnimalDevelopment (2d ed.) Wiley-Liss, New York, N.Y. Moreover, CX3Ckineexpression should serve to define certain cell subpopulations.

The CX3C chemokine producing profile of various cells is elucidatedherein. Screening a cDNA library generated from brain provided a novelcytokine, designated human CX3Ckine. Human CX3Ckine exhibits distantsimilarity to members of the C, C—C, and C—X—C chemokine families, withanother heretofore unrecognized number of amino acid residues separatingthe characteristic cysteines in the motif which is peculiar to andpartially defines chemokines. These observations suggest that theCX3Ckines represent novel additions to the chemokine superfamily.

CX3C chemokine protein biochemistry was assessed in mammalian expressionsystems. Human embryonic kidney 293 cells (HEK 293) transfected with amammalian expression construct encoding full-length CX3C chemokine weremetabolically labeled with ³⁵S cysteine and methionine. CX3C chemokinewas produced as a protein of Mr ˜95 kDa; control transfectedsupernatants contained no such species. Neuraminidase and glycosidasesreduced the Mr of CX3C chemokine from ˜95 kDa to ˜45 kDa, suggestingthat the recombinant form, is glycosylated substantially. CX3C chemokinecDNA, encoding a predicted membrane-bound protein, encodes aglycoprotein which is released from cells by an undefined mechanism.

The pro-migratory activities of CX3C chemokine have been assessed inmicrochemotaxis assays. CX3C chemokine appears to be a potent attractantof peripheral blood monocytes and T cells. Pro-migratory activity forblood neutrophils has been difficult to demonstrate.

The CX3C chemokine gene has been mapped to human chromosome 16. Mappingstudies also indicate the possibility of a pseudogene or related gene onhuman chromosome 14. Sequencing of genomic DNA fragments suggests CX3Cchemokine gene has an intron which begins near or in the codon encodingIle 64. Other intron/exon boundaries have yet to be mapped, but suchwill be easily accomplished by standard methods.

The membrane bound form of CX3Ckine possesses proadherent properties forcirculating T cells and monocytes. A secreted or soluble form,consisting of the chemokine domain and the stalk region, is able toinhibit this proadhesive activity. This suggests that the membrane boundform of CX3Ckine may be a potent regulator of circulating leukocytes,and thus may be involved in various inflammatory diseases, e.g.,arthritis. The soluble form may be used as a regulator of proadherence,especially in conditions of compromised immune response.

CX3C chemokine's properties as a T cell and monocyte chemoattractant,coupled with its distribution in brain and other organs, suggests thatCX3C chemokine may be involved in the pathogenesis of such CNSinflammatory disorders as multiple sclerosis, and other pathologiesinvolving neurogenic inflammation. Since CX3C chemokine distribution isnot limited to the brain, however, the entire spectrum of inflammatory,infectious, and immunoregulatory states thought to involve otherchemokines must also now be considered for CX3C chemokine. See, e.g.,Frank, Et al. (eds.) (1995) Samter's Immunologic Diseases 5th ed., vols.I and II, Little, Brown, and Co., Boston, Mass.

II. Definitions

The term “binding composition” refers to molecules that bind withspecificity to a CX3Ckine, e.g., in an antibody-antigen interaction.However, other compounds, e.g., receptor proteins, may also specificallyassociate with CX3Ckines to the exclusion of other molecules. Typically,the association will be in a natural physiologically relevantprotein-protein interaction, either covalent or non-covalent, and mayinclude members of a multiprotein complex, including carrier compoundsor dimerization partners. The molecule may be a polymer, or chemicalreagent. No implication as to whether a CX3Ckine is either the ligand orthe receptor of a ligand-receptor interaction is necessarilyrepresented, other than whether the interaction exhibits similarspecificity, e.g., specific affinity. A functional analog may be aligand with structural modifications, or may be a wholly unrelatedmolecule, e.g., which has a molecular shape which interacts with theappropriate ligand binding determinants. The ligands may serve asagonists or antagonists of the receptor, see, e.g., Goodman, et al.(eds.) (1990) Goodman & Gilman's: The Pharmacological Bases ofTherapeutics (8th ed.) Pergamon Press, Tarrytown, N.Y.

The term “binding agent:CX3Ckine protein complex”, as used herein,refers to a complex of a binding agent and a CX3Ckine protein that isformed by specific binding of the binding agent to the CX3Ckine protein,e.g., preferably the chemokine domain. Specific binding of the bindingagent means that the binding agent has a specific binding site thatrecognizes a site on the CX3Ckine protein. For example, antibodiesraised to a CX3Ckine protein and recognizing an epitope on the CX3Ckineprotein are capable of forming a binding agent:CX3Ckine protein complexby specific binding. Typically, the formation of a bindingagent:CX3Ckine protein complex allows the measurement of CX3Ckineprotein in a mixture of other proteins and biologics. The term“antibody:CX3Ckine protein complex” refers to an embodiment in which thebinding agent is an antibody. The antibody may be monoclonal,polyclonal, or a binding fragment of an antibody, e.g, an Fab of F(ab)₂fragment. The antibody will preferably be a polyclonal antibody forcross-reactivity purposes.

“Homologous” nucleic acid sequences, when compared, exhibit significantsimilarity. The standards for homology in nucleic acids are eithermeasures for homology generally used in the art by sequence comparisonand/or phylogenetic relationship, or based upon hybridizationconditions. Hybridization conditions are described in greater detailbelow.

An “isolated” nucleic acid is a nucleic acid, e.g., an RNA, DNA, or amixed polymer, which is substantially separated from other biologiccomponents which naturally accompany a native sequence, e.g., proteinsand flanking genomic sequences from the originating species. The termembraces a nucleic acid sequence which has been removed from itsnaturally occurring environment, and includes recombinant or cloned DNAisolates and chemically synthesized analogs, or analogs biologicallysynthesized by heterologous systems. A substantially pure moleculeincludes isolated forms of the molecule. An isolated nucleic acid willusually contain homogeneous nucleic acid molecules, but will, in someembodiments, contain nucleic acids with minor sequence heterogeneity.This heterogeneity is typically found at the polymer ends or portionsnot critical to a desired biological function or activity.

As used herein, the term “CX3Ckine protein” shall encompass, when usedin a protein context, a protein having amino acid sequences,particularly from the chemokine motif portions, shown in SEQ ID NO: 2,4, 6, or 8, or a significant fragment of such a protein, e.g.,preferably the chemokine domain. The invention also embraces apolypeptide which exhibits similar structure to human or mouse CX3Ckine,e.g., which interacts with CX3Ckine specific binding components. Thesebinding components, e.g., antibodies, typically bind to a CX3Ckine withhigh affinity, e.g., at least about 100 nM, usually better than about 30nM, preferably better than about 10 nM, and more preferably at betterthan about 3 nM.

The term “polypeptide” or “protein” as used herein includes asignificant fragment or segment of chemokine motif portion of aCX3Ckine, and encompasses a stretch of amino acid residues of at leastabout 8 amino acids, generally at least 10 amino acids, more generallyat least 12 amino acids, often at least 14 amino acids, more often atleast 16 amino acids, typically at least 18 amino acids, more typicallyat least 20 amino acids, usually at least 22 amino acids, more usuallyat least 24 amino acids, preferably at least 26 amino acids, morepreferably at least 28 amino acids, and, in particularly preferredembodiments, at least about 30 or more amino acids, e.g., 35, 40, 45,50, 60, 70, 80, etc.

A “recombinant” nucleic acid is defined either by its method ofproduction or its structure. In reference to its method of production,e.g., a product made by a process, the process is use of recombinantnucleic acid techniques, e.g., involving human intervention in thenucleotide sequence, typically selection or production. Alternatively,it can be a nucleic acid made by generating a sequence comprising fusionof two fragments which are not naturally contiguous to each other, butis meant to exclude products of nature, e.g., naturally occurringmutants. Thus, for example, products made by transforming cells with anynon-naturally occurring vector is encompassed, as are nucleic acidscomprising sequence derived using any synthetic oligonucleotide process.Such is often done to replace a codon with a redundant codon encodingthe same or a conservative amino acid, while typically introducing orremoving a sequence recognition site. Alternatively, it is performed tojoin together nucleic acid segments of desired functions to generate asingle genetic entity comprising a desired combination of functions notfound in the commonly available natural forms. Restriction enzymerecognition sites are often the target of such artificial manipulations,but other site specific targets, e.g., promoters, DNA replication sites,regulation sequences, control sequences, or other useful features may beincorporated by design. A similar concept is intended for a recombinant,e.g., fusion, polypeptide. Specifically included are synthetic nucleicacids which, by genetic code redundancy, encode polypeptides similar tofragments of these antigens, and fusions of sequences from variousdifferent species variants. Mutation of protease cleavage sites may alsobe accomplished.

“Solubility” is reflected by sedimentation measured in Svedberg units,which are a measure of the sedimentation velocity of a molecule underparticular conditions. The determination of the sedimentation velocitywas classically performed in an analytical ultracentrifuge, but istypically now performed in a standard ultracentrifuge. See, Freifelder(1982) Physical Biochemistry (2d ed.) W.H. Freeman & Co., San Francisco,Calif.; and Cantor and Schimmel (1980) Biophysical Chemistry parts 1-3,W.H. Freeman & Co., San Francisco, Calif. As a crude determination, asample containing a putatively soluble polypeptide is spun in a standardfull sized ultracentrifuge at about 50K rpm for about 10 minutes, andsoluble molecules will remain in the supernatant. A soluble particle orpolypeptide will typically be less than about 30S, more typically lessthan about 15S, usually less than about 10S, more usually less thanabout 6S, and, in particular embodiments, preferably less than about 4S,and more preferably less than about 3S. Solubility of a polypeptide orfragment depends upon the environment and the polypeptide. Manyparameters affect polypeptide solubility, including temperature,electrolyte environment, size and molecular characteristics of thepolypeptide, and nature of the solvent. Typically, the temperature atwhich the polypeptide is used ranges from about 4° C. to about 65° C.Usually the temperature at use is greater than about 18° C. and moreusually greater than about 22° C. For diagnostic purposes, thetemperature will usually be about room temperature or warmer, but lessthan the denaturation temperature of components in the assay. Fortherapeutic purposes, the temperature will usually be body temperature,typically about 37° C. for humans, though under certain situations thetemperature may be raised or lowered in situ or in vitro.

The size and structure of the polypeptide should generally be evaluatedin a substantially stable state, and usually not in a denatured state.The polypeptide may be associated with other polypeptides in aquaternary structure, e.g., to confer solubility, or associated withlipids or detergents in a manner which approximates natural lipidbilayer interactions.

The solvent will usually be a biologically compatible buffer, of a typeused for preservation of biological activities, and will usuallyapproximate a physiological solvent. Usually the solvent will have aneutral pH, typically between about 5 and 10, and preferably about 7.5.On some occasions, a detergent will be added, typically a mildnon-denaturing one, e.g., CHS (cholesteryl hemisuccinate) or CHAPS(3-[3-cholamidopropyl)dimethyl-ammonio]-1-propane sulfonate), or a lowenough concentration as to avoid significant disruption of structural orphysiological properties of the protein.

“Substantially pure” in a protein context typically means that theprotein is isolated from other contaminating proteins, nucleic acids,and other biologicals derived from the original source organism. Purity,or “isolation” may be assayed by standard methods, and will ordinarilybe at least about 50% pure, more ordinarily at least about 60% pure,generally at least about 70% pure, more generally at least about 80%pure, often at least about 85% pure, more often at least about 90% pure,preferably at least about 95% pure, more preferably at least about 98%pure, and in most preferred embodiments, at least 99% pure. Similarconcepts apply, e.g., to antibodies or nucleic acids.

“Substantial similarity” in the nucleic acid sequence comparison contextmeans either that the segments, or their complementary strands, whencompared, are identical when optimally aligned, with appropriatenucleotide insertions or deletions, in at least about 50% of thenucleotides, generally at least 56%, more generally at least 59%,ordinarily at least 62%, more ordinarily at least 65%, often at least68%, more often at least 71%, typically at least 74%, more typically atleast 77%, usually at least 80%, more usually at least about 85%,preferably at least about 90%, more preferably at least about 95 to 98%or more, and in particular embodiments, as high at about 99% or more ofthe nucleotides. Alternatively, substantial similarity exists when thesegments will hybridize under selective hybridization conditions, to astrand, or its complement, typically using a sequence derived from SEQID NO: 1, 3, 5 or 7. Typically, selective hybridization will occur whenthere is at least about 55% similarity over a stretch of at least about30 nucleotides, preferably at least about 65% over a stretch of at leastabout 25 nucleotides, more preferably at least about 75%, and mostpreferably at least about 90% over about 20 nucleotides. See Kanehisa(1984) Nuc. Acids Res. 12:203-213. The length of similarity comparison,as described, may be over longer stretches, and in certain embodimentswill be over a stretch of at least about 17 nucleotides, usually atleast about 20 nucleotides, more usually at least about 24 nucleotides,typically at least about 28 nucleotides, more typically at least about40 nucleotides, preferably at least about 50 nucleotides, and morepreferably at least about 75 to 100 or more nucleotides, e.g., 150, 200,etc.

“Stringent conditions”, in referring to homology or substantialsimilarity in the hybridization context, will be stringent combinedconditions of salt, temperature, organic solvents, and other parameters,typically those controlled in hybridization reactions. The combinationof parameters is more important than the measure of any singleparameter. Stringent temperature conditions will usually includetemperatures in excess of about 30° C., more usually in excess of about37° C., typically in excess of about 45° C., more typically in excess ofabout 55° C., preferably in excess of about 65° C., and more preferablyin excess of about 70° C. Stringent salt conditions will ordinarily beless than about 1000 mM, usually less than about 500 mM, more usuallyless than about 400 mM, typically less than about 300 mM, preferablyless than about 200 mM, and more preferably less than about 150 mM. See,e.g., Wetmur and Davidson (1968) J. Mol. Biol. 31:349-370. A nucleicacid probe which binds to a target nucleic acid under stringentconditions is specific for said target nucleic acid. Such a probe istypically more than 11 nucleotides in length, and is sufficientlyidentical or complementary to a target nucleic acid over the regionspecified by the sequence of the probe to bind the target understringent hybridization conditions.

CX3Ckines from other mammalian species can be cloned and isolated bycross-species hybridization of closely related species. See, e.g.,below. Similarity may be relatively low between distantly relatedspecies, and thus hybridization of relatively closely related species isadvisable. Alternatively, preparation of an antibody preparation whichexhibits less species specificity may be useful in expression cloningapproaches.

The phrase “specifically binds to an antibody” or “specificallyimmunoreactive with”, when referring to a protein or peptide, refers toa binding reaction which is determinative of the presence of the proteinin the presence of a heterogeneous population of proteins and otherbiological components. Thus, under designated immunoassay conditions,the specified antibodies bind to a particular protein and do notsignificantly bind other proteins present in the sample. Specificbinding to an antibody under such conditions may require an antibodythat is selected for its specificity for a particular protein. Forexample, antibodies raised to the human CX3Ckine protein immunogen withthe amino acid sequence depicted in SEQ ID NO: 2, 4, 6, or 8 can beselected to obtain antibodies specifically immunoreactive with CX3Ckineproteins and not with other proteins. These antibodies recognizeproteins highly similar to the homologous mouse CX3Ckine protein.

III. Nucleic Acids

Human CX3Ckine is exemplary of a larger class of structurally andfunctionally related proteins. These soluble chemokine proteins willserve to transmit signals between different cell types. The preferredembodiments, as disclosed, will be useful in standard procedures toisolate genes from different individuals or other species, e.g., warmblooded animals, such as birds and mammals. Cross hybridization willallow isolation of related genes encoding proteins from individuals,strains, or species. A number of different approaches are available tosuccessfully isolate a suitable nucleic acid clone based upon theinformation provided herein. Southern blot hybridization studies canqualitatively determine the presence of homologous genes in human,monkey, rat, dog, cow, and rabbit genomes under specific hybridizationconditions.

Complementary sequences will also be used as probes or primers. Basedupon identification of the likely amino terminus, other peptides shouldbe particularly useful, e.g., coupled with anchored vector or poly-Acomplementary PCR techniques or with complementary DNA of otherpeptides. Moreover, reverse translation using the redundancy in thegenetic code may provide synthetic genes which may encode essentiallyidentical proteins often with a condo usage selection preferred forexpression in a given host cell.

Techniques for nucleic acid manipulation of genes encoding CX3Ckineproteins, such as subcloning nucleic acid sequences encodingpolypeptides into expression vectors, labelling probes, DNAhybridization, and the like are described generally in Sambrook, et al.(1989) Molecular Cloning: A Laboratory Manual (2nd ed.) Vol. 1-3, ColdSpring Harbor Laboratory, Cold Spring Harbor Press, NY, which isincorporated herein by reference. This manual is hereinafter referred toas “Sambrook, et al.”

There are various methods of isolating DNA sequences encoding CX3Ckineproteins. For example, DNA is isolated from a genomic or cDNA libraryusing labeled oligonucleotide probes having sequences identical orcomplementary to the sequences disclosed herein. Full-length probes maybe used, or oligonucleotide probes may be generated by comparison of thesequences disclosed. Such probes can be used directly in hybridizationassays to isolate DNA encoding CX3Ckine proteins, or primers can bedesigned, e.g., using flanking sequence, for use in amplificationtechniques such as PCR, for the isolation of DNA encoding CX3Ckineproteins.

To prepare a cDNA library, mRNA is isolated from cells which express aCX3Ckine protein. cDNA is prepared from the mRNA and ligated into arecombinant vector. The vector is transfected into a recombinant hostfor propagation, screening, and cloning. Methods for making andscreening cDNA libraries are well known. See Gubler and Hoffman (1983)Gene 25:263-269 and Sambrook, et al.

For a genomic library, the DNA can be extracted from tissue and eithermechanically sheared or enzymatically digested to yield fragments, e.g.,of about 12-20 kb. The fragments are then separated by gradientcentrifugation and cloned in bacteriophage lambda vectors. These vectorsand phage are packaged in vitro, as described in Sambrook, et al.Recombinant phage are analyzed by plaque hybridization as described inBenton and Davis (1977) Science 196:180-182. Colony hybridization iscarried out as generally described in e.g., Grunstein, et al. (1975)Proc. Natl. Acad. Sci. USA. 72:3961-3965.

DNA encoding a CX3Ckine protein can be identified in either cDNA orgenomic libraries by its ability to hybridize with the nucleic acidprobes described herein, e.g., in colony or plaque hybridization assays.The corresponding DNA regions are isolated by standard methods familiarto those of skill in the art. See, e.g., Sambrook, et al. Alternatively,sequence databases, e.g., GenBank, may be evaluated for similar orcorresponding sequences.

Various methods of amplifying target sequences, such as the polymerasechain reaction, can also be used to prepare DNA encoding CX3Ckineproteins. Polymerase chain reaction (PCR) technology is used to amplifysuch nucleic acid sequences directly from mRNA, from cDNA, and fromgenomic libraries or cDNA libraries. The isolated sequences encodingCX3Ckine proteins may also be used as templates for PCR amplification.

Typically, in PCR techniques, oligonucleotide primers complementary totwo 5′ regions in two strands of the DNA region to be amplified aresynthesized. The polymerase chain reaction is then carried out using thetwo opposite primers. See Innis, et al. (eds.) (1990) PCR Protocols: AGuide to Methods and Applications Academic Press, San Diego, Calif.Primers can be selected to amplify the entire regions encoding afull-length CX3Ckine protein or to amplify smaller DNA segments asdesired. Once such regions are PCR-amplified, they can be sequenced andoligonucleotide probes can be prepared from sequence obtained usingstandard techniques. These probes can then be used to isolate DNA'sencoding CX3Ckine proteins.

Oligonucleotides for use as probes are usually chemically synthesizedaccording to the solid phase phosphoramidite triester method firstdescribed by Beaucage and Carruthers (1983) Tetrahedron Lett.22(20):1859-1862, or using an automated synthesizer, as described inNeedham-VanDevanter, et al. (1984) Nucleic Acids Res. 12:6159-6168.Purification of oligonucleotides is performed, e.g., by nativeacrylamide gel electrophoresis or by anion-exchange HPLC as described inPearson and Regnier (1983) J. Chrom. 255:137-149. The sequence of thesynthetic oligonucleotide can be verified using, e.g., the chemicaldegradation method of Maxam, A. M. and Gilbert, W. in Grossman, L. andMoldave (eds.) (1980) Methods in Enzymology 65:499-560 Academic Press,New York.

An isolated nucleic acid encoding a human CX3Ckine protein wasidentified. The nucleotide sequence and corresponding open reading frameare provided in SEQ ID NO: 1 and 2; with further sequences provided inSEQ ID NO: 3 and 4. Correspondingly, a mouse sequence was identified andits nucleotide and corresponding open reading frame are provided as SEQID NO: 5-8.

These CX3Ckines exhibit limited similarity to portions of chemokines,particularly the chemokine domains. See, e.g., Matsushima and Oppenheim(1989) Cytokine 1:2-13; Oppenheim, et al. (1991) Ann. Rev. Immunol.9:617-648; Schall (1991) Cytokine 3:165-183; and Gronenborn and Clore(1991) Protein Engineering 4:263-269. In particular, the human CX3Ckineshows similarity to the C class of chemokines in the carboxyl-terminalportion, particularly with respect to length, and at the positionscorresponding, in the numbering of mature human sequence, to thecys-ala-asp-pro sequence at positions 50-53; and the trp-val atpositions 57-58. CX3Ckines have a much longer carboxyl terminal tailthan the members of the CC or CXC chemokine families, and this “stalk”region may play a role in chemokine presentation. Notably, the spacingof conserved cysteine residues in the CXC and CC families of chemokinesare absent in the human CX3Ckine embodiment. Other features ofcomparison are apparent between the CX3Ckine and chemokine families.See, e.g., Lodi, et al. (1994) Science 263:1762-1766. In particular,β-sheet and α-helix residues can be determined using, e.g., RASMOLprogram, see Sayle and Milner-White (1995) TIBS 20:374-376; orGronenberg, et al. (1991) Protein Engineering 4:263-269; and otherstructural features are defined in Lodi, et al. (1994) Science263:1762-1767. These secondary and tertiary features assist in definingfurther the C, CC, and CXC structural features, along with spacing ofappropriate cysteine residues.

Based upon the structural modeling and insights in the binding regionsof the chemokines, it is predicted that residues in the mature humanprotein, lacking a signal of 24 residues, 26 (his), 28 (gln), 40 (ile),42 (glu), 47 (arg) and 48 (leu) should be important for chemokinebinding to cells. Residues at the amino terminus are probably notinvolved in receptor binding or specificity.

Moreover, exon boundaries are predicted to correspond to proteinsegments including the signal sequence through about the second residue(his) in the mature protein; from there to about three residues past thethird cys (around the arg-ala); and from there to the end. The thirdexon appears to exhibit relatively high similarity to the otherchemokines. The second exon would probably be most characteristic of theCX3C chemokines, and would be the preferred segment to use to search forhomology in other variants, e.g, species or otherwise. In particular,segments expected to be preferred in producing CX3C chemokine specificantibodies will include peptides or sequence in the region from thesecond residue of the mature protein (his) through about the thirdresidue after the third cysteine (arg). Fragments of at least about 8-10residues in that region would be especially interesting peptides, e.g.,starting at residue positions of the mature 1, 2, 3, etc. Thosefragments would typically end in that region, e.g., at residue 37, 36,35, etc. Other interesting peptides of various lengths would includeones which begin or end in other positions of the protein, e.g., atresidues 87, 86, etc., with lengths ranging, e.g., from about 8 to 20,25, 30, 35, 40, etc. Corresponding fragments of other mammalianCX3Ckine, e.g., mouse, will be preferred embodiments.

This invention provides isolated DNA or fragments to encode a CX3Ckineprotein. In addition, this invention provides isolated or recombinantDNA which encodes a protein or polypeptide which is capable ofhybridizing under appropriate conditions, e.g., high stringency, withthe DNA sequences described herein. Said biologically active protein orpolypeptide can be an intact ligand, or fragment, and have an amino acidsequence as disclosed in SEQ ID NO: 2, 4, 6, or 8. Preferred embodimentswill be full length natural sequences, from isolates, e.g., about 11,000to 12,500 daltons in size when unglycosylated, or fragments of at leastabout 6,000 daltons, more preferably at least about 8,000 daltons. Inglycosylated form, the protein may exceed 12,500 daltons. Further, thisinvention contemplates the use of isolated or recombinant DNA, orfragments thereof, which encode proteins which are homologous to aCX3Ckine protein or which were isolated using cDNA encoding a CX3Ckineprotein as a probe. The isolated DNA can have the respective regulatorysequences in the 5′ and 3′ flanks, e.g., promoters, enhancers, poly-Aaddition signals, and others.

IV. Making CX3Ckines

DNAs which encode a CX3Ckine or fragments thereof can be obtained bychemical synthesis, screening cDNA libraries, or by screening genomiclibraries prepared from a wide variety of cell lines or tissue samples.The redundancy of the genetic code provides a number of polynucleotidesequences which should encode the same protein.

These DNAs can be expressed in a wide variety of host cells for thesynthesis of a full-length protein or fragments which can in turn, e.g.,be used to generate polyclonal or monoclonal antibodies; for bindingstudies; for construction and expression of modified molecules; and forstructure/function studies. Each CX3Ckine or its fragments, e.g., thechemokine domain, can be expressed in host cells that are transformed ortransfected with appropriate expression vectors. These molecules can besubstantially purified to be free of protein or cellular contaminants,other than those derived from the recombinant host, and therefore areparticularly useful in pharmaceutical compositions when combined with apharmaceutically acceptable carrier and/or diluent. The antigen, e.g.,CX3Ckine, or portions thereof, may be expressed as fusions with otherproteins or possessing an epitope tag. Such is applicable also toantigen binding sites.

Expression vectors are typically self-replicating DNA or RNA constructscontaining the desired antigen gene or its fragments, usually operablylinked to appropriate genetic control elements that are recognized in asuitable host cell. The specific type of control elements necessary toeffect expression will depend upon the eventual host cell used.Generally, the genetic control elements can include a prokaryoticpromoter system or a eukaryotic promoter expression control system, andtypically include a transcriptional promoter, an optional operator tocontrol the onset of transcription, transcription enhancers to elevatethe level of mRNA expression, a sequence that encodes a suitableribosome binding site, and sequences that terminate transcription andtranslation. Expression vectors also usually contain an origin ofreplication that allows the vector to replicate independently from thehost cell.

The vectors of this invention encompass DNAs which encode a CX3Ckine, ora fragment thereof, typically encoding, e.g., a biologically activepolypeptide, or protein. The DNA can be under the control of a viralpromoter and can encode a selection marker. This invention furthercontemplates use of such expression vectors which are capable ofexpressing eukaryotic cDNA coding for a CX3Ckine protein in aprokaryotic or eukaryotic host, where the vector is compatible with thehost and where the eukaryotic cDNA coding for the protein is insertedinto the vector such that growth of the host containing the vectorexpresses the cDNA in question. Usually, expression vectors are designedfor stable replication in their host cells or for amplification togreatly increase the total number of copies of the desirable gene percell. It is not always necessary to require that an expression vectorreplicate in a host cell, e.g., it is possible to effect transientexpression of the protein or its fragments in various hosts usingvectors that do not contain a replication origin that is recognized bythe host cell. It is also possible to use vectors that cause integrationof a CX3Ckine gene or its fragments into the host DNA by recombination,or to integrate a promoter which controls expression of an endogenousgene.

Vectors, as used herein, contemplate plasmids, viruses, bacteriophage,integratable DNA fragments, and other vehicles which enable theintegration of DNA fragments into the genome of the host. Expressionvectors are specialized vectors which contain genetic control elementsthat effect expression of operably linked genes. Plasmids are the mostcommonly used form of vector, but many other forms of vectors whichserve an equivalent function are suitable for use herein. See, e.g.,Pouwels, et al. (1985 and Supplements) Cloning Vectors: A LaboratoryManual Elsevier, N.Y.; and Rodriquez, et al. (eds.) (1988) Vectors: ASurvey of Molecular Cloning Vectors and Their Uses Buttersworth, Boston,Mass.

Suitable host cells include prokaryotes, lower eukaryotes, and highereukaryotes. Prokaryotes include both gram negative and gram positiveorganisms, e.g., E. coli and B. subtilis. Lower eukaryotes includeyeasts, e.g., S. cerevisiae and Pichia, and species of the genusDictyostelium. Higher eukaryotes include established tissue culture celllines from animal cells, both of non-mammalian origin, e.g., insectcells, and birds, and of mammalian origin, e.g., human, primates, androdents.

Prokaryotic host-vector systems include a wide variety of vectors formany different species. As used herein, E. coli and its vectors will beused generically to include equivalent vectors used in otherprokaryotes. A representative vector for amplifying DNA is pBR322 or itsderivatives. Vectors that can be used to express CX3Ckines or CX3Ckinefragments include, but are not limited to, such vectors as thosecontaining the lac promoter (pUC-series); trp promoter (pBR322-trp); Ipppromoter (the pIN-series); lambda-pP or pR promoters (pOTS); or hybridpromoters such as ptac (pDR540). See Brosius, et al. (1988) “ExpressionVectors Employing Lambda-, trp-, lac-, and Ipp-derived Promoters”, inRodriguez and Denhardt (eds.) Vectors: A Survey of Molecular CloningVectors and Their Uses 10:205-236 Buttersworth, Boston, Mass.

Lower eukaryotes, e.g., yeasts and Dictyostelium, may be transformedwith CX3Ckine sequence containing vectors. For purposes of thisinvention, the most common lower eukaryotic host is the baker's yeast,Saccharomyces cerevisiae. It will be used generically to represent lowereukaryotes although a number of other strains and species are alsoavailable. Yeast vectors typically consist of a replication origin(unless of the integrating type), a selection gene, a promoter, DNAencoding the desired protein or its fragments, and sequences fortranslation termination, polyadenylation, and transcription termination.Suitable expression vectors for yeast include such constitutivepromoters as 3-phosphoglycerate kinase and various other glycolyticenzyme gene promoters or such inducible promoters as the alcoholdehydrogenase 2 promoter or metallothionine promoter. Suitable vectorsinclude derivatives of the following types: self-replicating low copynumber (such as the YRp-series), self-replicating high copy number (suchas the YEp-series); integrating types (such as the YIp-series), ormini-chromosomes (such as the YCp-series).

Higher eukaryotic tissue culture cells are typically the preferred hostcells for expression of the functionally active CX3Ckine protein. Inprinciple, many higher eukaryotic tissue culture cell lines may be used,e.g., insect baculovirus expression systems, whether from aninvertebrate or vertebrate source. However, mammalian cells arepreferred to achieve proper processing, both cotranslationally andposttranslationally. Transformation or transfection and propagation ofsuch cells is routine. Useful cell lines include HeLa cells, Chinesehamster ovary (CHO) cell lines, baby rat kidney (BRK) cell lines, insectcell lines, bird cell lines, and monkey (COS) cell lines. Expressionvectors for such cell lines usually include an origin of replication, apromoter, a translation initiation site, RNA splice sites (e.g., ifgenomic DNA is used), a polyadenylation site, and a transcriptiontermination site. These vectors also may contain a selection gene oramplification gene. Suitable expression vectors may be plasmids,viruses, or retroviruses carrying promoters derived, e.g., from suchsources as from adenovirus, SV40, parvoviruses, vaccinia virus, orcytomegalovirus. Representative examples of suitable expression vectorsinclude pcDNA1; pCD, see Okayama, et al. (1985) Mol. Cell. Biol.5:1136-1142; pMC1neo Poly-A, see Thomas, et al. (1987) Cell 51:503-512;and a baculovirus vector such as pAC 373 or pAC 610.

It is likely that CX3Ckines need not be glycosylated to elicitbiological responses. However, it will occasionally be desirable toexpress a CX3Ckine polypeptide in a system which provides a specific ordefined glycosylation pattern. In this case, the usual pattern will bethat provided naturally by the expression system. However, the patternwill be modifiable by exposing the polypeptide, e.g., in unglycosylatedform, to appropriate glycosylating proteins introduced into aheterologous expression system. For example, the CX3Ckine gene may beco-transformed with one or more genes encoding mammalian or otherglycosylating enzymes. It is further understood that over glycosylationmay be detrimental to CX3Ckine biological activity, and that one ofskill may perform routine testing to optimize the degree ofglycosylation which confers optimal biological activity.

A CX3Ckine, or a fragment thereof, may be engineered to be phosphatidylinositol (PI) linked to a cell membrane, but can be removed frommembranes by treatment with a phosphatidyl inositol cleaving enzyme,e.g., phosphatidyl inositol phospholipase-C. This releases the antigenin a biologically active form, and allows purification by standardprocedures of protein chemistry. See, e.g., Low (1989) Biochem. Biophys.Acta 988:427-454; Tse, et al. (1985) Science 230:1003-1008; and Brunner,et al. (1991) J. Cell Biol. 114:1275-1283.

Now that CX3Ckines have been characterized, fragments or derivativesthereof can be prepared by conventional processes for synthesizingpeptides. These include processes such as are described in Stewart andYoung (1984) Solid Phase Peptide Synthesis Pierce Chemical Co.,Rockford, Ill.; Bodanszky and Bodanszky (1984) The Practice of PeptideSynthesis Springer-Verlag, New York, N.Y.; and Bodanszky (1984) ThePrinciples of Peptide Synthesis Springer-Verlag, New York, N.Y. Forexample, an azide process, an acid chloride process, an acid anhydrideprocess, a mixed anhydride process, an active ester process (forexample, p-nitrophenyl ester, N-hydroxysuccinimide ester, or cyanomethylester), a carbodiimidazole process, an oxidative-reductive process, or adicyclohexylcarbodiimide (DCCD)/additive process can be used. Solidphase and solution phase syntheses are both applicable to the foregoingprocesses.

The prepared protein and fragments thereof can be isolated and purifiedfrom the reaction mixture by means of peptide separation, for example,by extraction, precipitation, electrophoresis and various forms ofchromatography, and the like. The CX3Ckines of this invention can beobtained in varying degrees of purity depending upon its desired use.Purification can be accomplished by use of known protein purificationtechniques or by the use of the antibodies or binding partners hereindescribed, e.g., in immunoabsorbant affinity chromatography. See, e.g.,Coligan, et al. (eds.) (1995 and periodic supplements) Current Protocolsin Protein Science, John Wiley and Sons, New York, N.Y. Thisimmunoabsorbant affinity chromatography is carried out by first linkingthe antibodies to a solid support and then contacting the linkedantibodies with solubilized lysates of appropriate source cells, lysatesof other cells expressing the ligand, or lysates or supernatants ofcells producing the CX3Ckines as a result of recombinant DNA techniques,see below.

Multiple cell lines may be screened for one which expresses a CX3Ckineat a high level compared with other cells. Various cell lines, e.g., amouse thymic stromal cell line TA4, is screened and selected for itsfavorable handling properties. Natural CX3Ckines can be isolated fromnatural sources, or by expression from a transformed cell using anappropriate expression vector. Purification of the expressed protein isachieved by standard procedures, or may be combined with engineeredmeans for effective purification at high efficiency from cell lysates orsupernatants. Epitope or other tags, e.g., FLAG or His₆ segments, can beused for such purification features.

V. Antibodies

Antibodies can be raised to various CX3Ckines, including individual,polymorphic, allelic, strain, or species variants, and fragmentsthereof, both in their naturally occurring (full-length) forms and intheir recombinant forms. Additionally, antibodies can be raised toCX3Ckines in either their active or native forms or in their inactive ordenatured forms. Anti-idiotypic antibodies may also be used.

A. Antibody Production

A number of immunogens may be used to produce antibodies specificallyreactive with CX3Ckine proteins. Recombinant protein is a preferredimmunogen for the production of monoclonal or polyclonal antibodies.Naturally occurring protein may also be used either in pure or impureform. Synthetic peptides, made using the human or mouse CX3Ckine proteinsequences described herein, may also used as an immunogen for theproduction of antibodies to CX3Ckines, e.g., the chemokine domainsthereof. Recombinant protein can be expressed in eukaryotic orprokaryotic cells as described herein, and purified as described.Naturally folded or denatured material can be used, as appropriate, forproducing antibodies. Either monoclonal or polyclonal antibodies may begenerated for subsequent use in immunoassays to measure the protein.

Methods of producing polyclonal antibodies are known to those of skillin the art. Typically, an immunogen, preferably a purified protein, ismixed with an adjuvant and animals are immunized with the mixture. Theanimal's immune response to the immunogen preparation is monitored bytaking test bleeds and determining the titer of reactivity to theCX3Ckine protein or fragment of interest. When appropriately high titersof antibody to the immunogen are obtained, usually after repeatedimmunizations, blood is collected from the animal and antisera areprepared. Further fractionation of the antisera to enrich for antibodiesreactive to the protein can be done if desired. See, e.g., Harlow andLane; or Coligan.

Monoclonal antibodies may be obtained by various techniques familiar tothose skilled in the art. Typically, spleen cells from an animalimmunized with a desired antigen are immortalized, commonly by fusionwith a myeloma cell (see, Kohler and Milstein (1976) Eur. J. Immunol.6:511-519, incorporated herein by reference). Alternative methods ofimmortalization include transformation with Epstein Barr Virus,oncogenes, or retroviruses, or other methods known in the art. Coloniesarising from single immortalized cells are screened for production ofantibodies of the desired specificity and affinity for the antigen, andyield of the monoclonal antibodies produced by such cells may beenhanced by various techniques, including injection into the peritonealcavity of a vertebrate host. Alternatively, one may isolate DNAsequences which encode a monoclonal antibody or a binding fragmentthereof by screening a DNA library from human B cells according, e.g.,to the general protocol outlined by Huse, et al. (1989) Science246:1275-1281.

Antibodies, including binding fragments and single chain versions,against predetermined fragments of CX3Ckines can be raised byimmunization of animals with conjugates of the fragments with carrierproteins as described above. Monoclonal antibodies are prepared fromcells secreting the desired antibody. These antibodies can be screenedfor binding to normal or defective CX3Ckines, or screened for agonisticor antagonistic activity, e.g., mediated through a receptor. Thesemonoclonal antibodies will usually bind with at least a K_(D) of about 1mM, more usually at least about 300 μM, typically at least about 10 μM,more typically at least about 30 μM, preferably at least about 10 μM,and more preferably at least about 3 μM or better.

In some instances, it is desirable to prepare monoclonal antibodies fromvarious mammalian hosts, such as mice, rodents, primates, humans, etc.Description of techniques for preparing such monoclonal antibodies maybe found in, e.g., Stites, et al. (eds.) Basic and Clinical Immunology(4th ed.) Lange Medical Publications, Los Altos, Calif., and referencescited therein; Harlow and Lane (1988) Antibodies: A Laboratory ManualCSH Press; Goding (1986) Monoclonal Antibodies: Principles and Practice(2d ed.) Academic Press, New York, N.Y.; and particularly in Kohler andMilstein (1975) Nature 256:495-497, which discusses one method ofgenerating monoclonal antibodies. Summarized briefly, this methodinvolves injecting an animal with an immunogen. The animal is thensacrificed and cells taken from its spleen, which are then fused withmyeloma cells. The result is a hybrid cell or “hybridoma” that iscapable of reproducing in vitro. The population of hybridomas is thenscreened to isolate individual clones, each of which secrete a singleantibody species to the immunogen. In this manner, the individualantibody species obtained are the products of immortalized and clonedsingle B cells from the immune animal generated in response to aspecific site recognized on the immunogenic substance.

Other suitable techniques involve selection of libraries of antibodiesin phage or similar vectors. See, e.g., Huse, et al. (1989) “Generationof a Large Combinatorial Library of the Immunoglobulin Repertoire inPhage Lambda,” Science 246:1275-1281; and Ward, et al. (1989) Nature341:544-546. The polypeptides and antibodies of the present inventionmay be used with or without modification, including chimeric orhumanized antibodies. Frequently, the polypeptides and antibodies willbe labeled by joining, either covalently or non-covalently, a substancewhich provides for a detectable signal. A wide variety of labels andconjugation techniques are known and are reported extensively in boththe scientific and patent literature. Suitable labels includeradionuclides, enzymes, substrates, cofactors, inhibitors, fluorescentmoieties, chemiluminescent moieties, magnetic particles, and the like.Patents, teaching the use of such labels include U.S. Pat. Nos.3,817,837; 3,850,752; 3,939,350; 3,996,345; 4,277,437; 4,275,149; and4,366,241. Also, recombinant immunoglobulins may be produced. See,Cabilly, U.S. Pat. No. 4,816,567; and Queen, et al. (1989) Proc. Nat'lAcad. Sci. USA 86:10029-10033.

The antibodies of this invention are useful for affinity chromatographyin isolating CX3Ckine protein. Columns can be prepared where theantibodies are linked to a solid support, e.g., particles, such asagarose, SEPHADEX, or the like, where a cell lysate or supernatant maybe passed through the column, the column washed, followed by increasingconcentrations of a mild denaturant, whereby purified CX3Ckine proteinwill be released.

The antibodies may also be used to screen expression libraries forparticular expression products. Usually the antibodies used in such aprocedure will be labeled with a moiety allowing easy detection ofpresence of antigen by antibody binding.

Antibodies to CX3Ckines may be used for the identification of cellpopulations expressing CX3Ckines. By assaying the expression products ofcells expressing CX3Ckines it is possible to diagnose disease, e.g.,immune-compromised conditions.

Antibodies raised against each CX3Ckine will also be useful to raiseanti-idiotypic antibodies. These will be useful in detecting ordiagnosing various immunological conditions related to expression of therespective antigens.

B. Immunoassays

A particular protein can be measured by a variety of immunoassaymethods. For a review of immunological and immunoassay procedures ingeneral, see Stites and Terr (eds.) (1991) Basic and Clinical Immunology(7th ed.). Moreover, the immunoassays of the present invention can beperformed in many configurations, which are reviewed extensively inMaggio (ed.) (1980) Enzyme Immunoassay CRC Press, Boca Raton, Fla.;Tijan (1985) “Practice and Theory of Enzyme Immunoassays,” LaboratoryTechniques in Biochemistry and Molecular Biology, Elsevier SciencePublishers B.V., Amsterdam; and Harlow and Lane Antibodies, A LaboratoryManual, supra, each of which is incorporated herein by reference. Seealso Chan (ed.) (1987) Immunoassay: A Practical Guide Academic Press,Orlando, Fla.; Price and Newman (eds.) (1991) Principles and Practice ofImmunoassays Stockton Press, NY; and Ngo (ed.) (1988) Non-isotopicImmunoassays Plenum Press, NY.

Immunoassays for measurement of CX3Ckine proteins can be performed by avariety of methods known to those skilled in the art. In brief,immunoassays to measure the protein can be competitive or noncompetitivebinding assays. In competitive binding assays, the sample to be analyzedcompetes with a labeled analyte for specific binding sites on a captureagent bound to a solid surface. Preferably the capture agent is anantibody specifically reactive with CX3Ckine proteins produced asdescribed above. The concentration of labeled analyte bound to thecapture agent is inversely proportional to the amount of free analytepresent in the sample.

In a competitive binding immunoassay, the CX3Ckine protein present inthe sample competes with labeled protein for binding to a specificbinding agent, for example, an antibody specifically reactive with theCX3Ckine protein. The binding agent may be bound to a solid surface toeffect separation of bound labeled protein from the unbound labeledprotein. Alternatively, the competitive binding assay may be conductedin liquid phase and a variety of techniques known in the art may be usedto separate the bound labelled protein from the unbound labeled protein.Following separation, the amount of bound labeled protein is determined.The amount of protein present in the sample is inversely proportional tothe amount of labeled protein binding.

Alternatively, a homogeneous immunoassay may be performed in which aseparation step is not needed. In these immunoassays, the label on theprotein is altered by the binding of the protein to its specific bindingagent. This alteration in the labeled protein results in a decrease orincrease in the signal emitted by label, so that measurement of thelabel at the end of the immunoassay allows for detection or quantitationof the protein.

CX3Ckine proteins may also be determined by a variety of noncompetitiveimmunoassay methods. For example, a two-site, solid phase sandwichimmunoassay may be used. In this type of assay, a binding agent for theprotein, for example an antibody, is attached to a solid support. Asecond protein binding agent, which may also be an antibody, and whichbinds the protein at a different site, is labelled. After binding atboth sites on the protein has occurred, the unbound labeled bindingagent is removed and the amount of labeled binding agent bound to thesolid phase is measured. The amount of labeled binding agent bound isdirectly proportional to the amount of protein in the sample.

Western blot analysis can be used to determine the presence of CX3Ckineproteins in a sample. Electrophoresis is carried out, for example, on atissue sample suspected of containing the protein. Followingelectrophoresis to separate the proteins, and transfer of the proteinsto a suitable solid support, e.g., a nitrocellulose filter, the solidsupport is incubated with an antibody reactive with the protein. Thisantibody may be labeled, or alternatively may be detected by subsequentincubation with a second labeled antibody that binds the primaryantibody.

The immunoassay formats described above employ labeled assay components.The label may be coupled directly or indirectly to the desired componentof the assay according to methods well known in the art. A wide varietyof labels and methods may be used. Traditionally, a radioactive labelincorporating ³H, ¹²⁵I, ³⁵S, ¹⁴C, or ³²P was used. Non-radioactivelabels include ligands which bind to labeled antibodies, fluorophores,chemiluminescent agents, enzymes, and antibodies which can serve asspecific binding pair members for a labeled ligand. The choice of labeldepends on sensitivity required, ease of conjugation with the compound,stability requirements, and available instrumentation. For a review ofvarious labelling or signal producing systems which may be used, seeU.S. Pat. No. 4,391,904, which is incorporated herein by reference.

Antibodies reactive with a particular protein can also be measured by avariety of immunoassay methods. For a review of immunological andimmunoassay procedures applicable to the measurement of antibodies byimmunoassay techniques, see Stites and Terr (eds.) Basic and ClinicalImmunology (7th ed.) supra; Maggio (ed.) Enzyme Immunoassay, supra; andHarlow and Lane Antibodies, A Laboratory Manual, supra.

In brief, immunoassays to measure antisera reactive with CX3Ckineproteins can be competitive or noncompetitive binding assays. Incompetitive binding assays, the sample analyte competes with a labeledanalyte for specific binding sites on a capture agent bound to a solidsurface. Preferably the capture agent is a purified recombinant CX3Ckineprotein produced as described above. Other sources of CX3Ckine proteins,including isolated or partially purified naturally occurring protein,may also be used. Noncompetitive assays include sandwich assays, inwhich the sample analyte is bound between two analyte-specific bindingreagents. One of the binding agents is used as a capture agent and isbound to a solid surface. The second binding agent is labeled and isused to measure or detect the resultant complex by visual or instrumentmeans. A number of combinations of capture agent and labelled bindingagent can be used. A variety of different immunoassay formats,separation techniques, and labels can be also be used similar to thosedescribed above for the measurement of CX3Ckine proteins.

VI. Purified CX3Ckines

Human CX3Ckine amino acid sequences are provided in SEQ ID NO: 2 and 4.Mouse nucleotide and amino acid sequences are provided in SEQ ID NO: 5,6, 7, and 8.

Purified protein or defined peptides are useful for generatingantibodies by standard methods, as described above. Synthetic peptidesor purified protein, e.g., the chemokine domains, can be presented to animmune system to generate polyclonal and monoclonal antibodies. See,e.g., Coligan (1991) Current Protocols in Immunology Wiley/Greene, NY;and Harlow and Lane (1989) Antibodies: A Laboratory Manual Cold SpringHarbor Press, NY, which are incorporated herein by reference.Alternatively, a CX3Ckine receptor can be useful as a specific bindingreagent, and advantage can be taken of its specificity of binding, for,e.g., purification of a CX3Ckine ligand.

The specific binding composition can be used for screening an expressionlibrary made from a cell line which expresses a CX3Ckine. Many methodsfor screening are available, e.g., standard staining of surfaceexpressed ligand, or by panning. Screening of intracellular expressioncan also be performed by various staining or immunofluorescenceprocedures. The binding compositions could be used to affinity purify orsort out cells expressing the ligand.

The peptide segments, along with comparison to homologous genes, canalso be used to produce appropriate oligonucleotides to screen alibrary. The genetic code can be used to select appropriateoligonucleotides useful as probes for screening. In combination withpolymerase chain reaction (PCR) techniques, synthetic oligonucleotideswill be useful in selecting desired clones from a library, includingnatural allelic an polymorphic variants.

The peptide sequences allow preparation of peptides to generateantibodies to recognize such segments, and allow preparation ofoligonucleotides which encode such sequences. The sequence also allowsfor synthetic preparation, e.g., see Dawson, et al. (1994) Science266:776-779. Since CX3Ckines appear to be soluble proteins, the genewill normally possess an N-terminal signal sequence, which is removedupon processing and secretion, and the putative cleavage site is betweenamino acids 24 (gly) and 25 (gin) in SEQ ID NO: 2 or 4, though it may beslightly in either direction. Analysis of the structural features incomparison with the most closely related reported sequences has revealedsimilarities with other cytokines, particularly the class of proteinsknown as chemokines. Within the chemokines are two subgroups, the CC andCXC subgroups. The CX3Ckine family shares various features with each ofthese groups, but its combination of features is distinctive and definesa new family of related chemokines.

While further structural features result from the sequences provided inSEQ ID NO: 1 through 8, the “chemokine on a stick” feature is providedthrough the stalk region which possesses many sites which may provide aheavily glycosylated domain. The stalk structure may be important inCX3C chemokine presentation to other cells. In fact, it appears that thestalk region may be processed to release the soluble chemokine. Thissuggests the possibility of substituting the CX3C chemokine domain withother chemokines to effect efficient presentation to appropriate targetcells.

In addition, the “stalk” regions are likely to affect solubility andpharmacological aspects of the protein. As such, this region will be thetarget of analysis to evaluate and modulate such features aspharmacokinetics. Truncation of that portion may affect half-life,clearance, and accessibility of the chemokine domains.

VII. Physical Variants

This invention also encompasses proteins or peptides having substantialamino acid sequence similarity with an amino acid sequence of aCX3Ckine. Natural variants include individual, polymorphic, allelic,strain, or species variants.

Amino acid sequence similarity, or sequence identity, is determined byoptimizing residue matches, if necessary, by introducing gaps asrequired. This changes when considering conservative substitutions asmatches. Conservative substitutions typically include substitutionswithin the following groups: glycine, alanine; valine, isoleucine,leucine; aspartic acid, glutamic acid; asparagine, glutamine; serine,threonine; lysine, arginine; and phenylalanine, tyrosine. Homologousamino acid sequences include natural polymorphic, allelic, andinterspecies variations in each respective protein sequence. Typicalhomologous proteins or peptides will have from 50-100% similarity (ifgaps can be introduced), to 75-100% similarity (if conservativesubstitutions are included) with the amino acid sequence of theCX3Ckine. Similarity measures will be at least about 50%, generally atleast 60%, more generally at least 65%, usually at least 70%, moreusually at least 75%, preferably at least 80%, and more preferably atleast 80%, and in particularly preferred embodiments, at least 85% ormore. See also Needleham, et al. (1970) J. Mol. Biol. 48:443-453;Sankoff, et al. (1983) Time Warps, String Edits, and Macromolecules: TheTheory and Practice of Sequence Comparison Chapter One, Addison-Wesley,Reading, Mass.; and software packages from IntelliGenetics, MountainView, Calif.; and the University of Wisconsin Genetics Computer Group,Madison, Wis.

Nucleic acids encoding mammalian CX3Ckine proteins will typicallyhybridize to the nucleic acid sequence of SEQ ID NO: 1, 3, 5 or 7 understringent conditions. For example, nucleic acids encoding human CX3Ckineproteins will normally hybridize to the nucleic acid of SEQ ID NO: 1under stringent hybridization conditions. Generally, stringentconditions are selected to be about 10° C. lower than the thermalmelting point (Tm) for the probe sequence at a defined ionic strengthand pH. The Tm is the temperature (under defined ionic strength and pH)at which 50% of the target sequence hybridizes to a perfectly matchedprobe. Typically, stringent conditions will be those in which the saltconcentration is about 0.2 molar at pH 7 and the temperature is at leastabout 50° C. Other factors may significantly affect the stringency ofhybridization, including, among others, base composition and size of thecomplementary strands, the presence of organic solvents such asformamide, and the extent of base mismatching. A preferred embodimentwill include nucleic acids which will bind to disclosed sequences in 50%formamide and 200 mM NaCl at 42° C.

An isolated CX3Ckine DNA can be readily modified by nucleotidesubstitutions, nucleotide deletions, nucleotide insertions, and shortinversions of nucleotide stretches. These modifications result in novelDNA sequences which encode CX3Ckine antigens, their derivatives, orproteins having highly similar physiological, immunogenic, or antigenicactivity.

Modified sequences can be used to produce mutant antigens or to enhanceexpression. Enhanced expression may involve gene amplification,increased transcription, increased translation, and other mechanisms.Such mutant CX3Ckine derivatives include predetermined or site-specificmutations of the respective protein or its fragments. “Mutant CX3Ckine”encompasses a polypeptide otherwise falling within the homologydefinition of the human CX3Ckine as set forth above, but having an aminoacid sequence which differs from that of a CX3Ckine as found in nature,whether by way of deletion, substitution, or insertion. In particular,“site specific mutant CX3Ckine” generally includes proteins havingsignificant similarity with a protein having a sequence of SEQ ID NO: 2,4, 6, or 8, and as sharing various biological activities, e.g.,antigenic or immunogenic, with those sequences, and in preferredembodiments contain most or all of the disclosed sequence. This appliesalso to polymorphic variants from different individuals. Similarconcepts apply to different CX3Ckine proteins, particularly those foundin various warm blooded animals, e.g., mammals and birds. As statedbefore, it is emphasized that descriptions are generally meant toencompass other CX3Ckine proteins, not limited to the human or mouseembodiments specifically discussed.

Although site specific mutation sites are predetermined, mutants neednot be site specific. CX3Ckine mutagenesis can be conducted by makingamino acid insertions or deletions. Substitutions, deletions,insertions, or any combinations may be generated to arrive at a finalconstruct. These include amino acid residue substitution levels fromnone, one, two, three, five, seven, ten, twelve, fifteen, etc.Insertions include amino- or carboxyl-terminal fusions, e.g. epitopetags. Random mutagenesis can be conducted at a target codon and theexpressed mutants can then be screened for the desired activity. Methodsfor making substitution mutations at predetermined sites in DNA having aknown sequence are well known in the art, e.g., by M13 primermutagenesis or polymerase chain reaction (PCR) techniques. See also,Sambrook, et al. (1989) and Ausubel, et al. (1987 and Supplements). Themutations in the DNA normally should not place coding sequences out ofreading frames and preferably will not create complementary regions thatcould hybridize to produce secondary mRNA structure such as loops orhairpins.

The present invention also provides recombinant proteins, e.g.,heterologous fusion proteins using segments from these proteins, boththe CX3Ckine, or antigen binding sites. A heterologous fusion protein isa fusion of proteins or segments which are naturally not normally fusedin the same manner. Thus, the fusion product of an immunoglobulin with aCX3Ckine polypeptide is a continuous protein molecule having sequencesfused in a typical peptide linkage, typically made as a singletranslation product and exhibiting properties derived from each sourcepeptide. A similar concept applies to heterologous nucleic acidsequences.

In addition, new constructs may be made from combining similarfunctional domains from other proteins. For example, protein-binding orother segments may be “swapped” between different new fusionpolypeptides or fragments. See, e.g., Cunningham, et al. (1989) Science243:1330-1336; and O'Dowd, et al. (1988) J. Biol. Chem. 263:15985-15992.Thus, new chimeric polypeptides exhibiting new combinations ofspecificities will result from the functional linkage of protein-bindingspecificities and other functional domains.

VIII. Binding Agent:CX3Ckine Protein Complexes

A CX3Ckine protein that specifically binds to or that is specificallyimmunoreactive with an antibody generated against a defined immunogen,such as an immunogen consisting of the amino acid sequence of SEQ ID NO:2. 4, 6, or 8, is typically determined in an immunoassay. Theimmunoassay uses a polyclonal antiserum which was raised to a protein ofSEQ ID NO: 2. 4, 6, or 8. This antiserum is selected to have lowcrossreactivity against other chemokines and any such crossreactivity isremoved by immunoabsorbtion prior to use in the immunoassay.

In order to produce antisera for use in an immunoassay, the protein ofSEQ ID NO: 2. 4, 6, or 8, is isolated as described herein. For example,recombinant protein may be produced in a mammalian cell line. An inbredstrain of mice such as balb/c is immunized with the protein of SEQ IDNO: 2. 4, 6, or 8, using a standard adjuvant, such as Freund's adjuvant,and a standard mouse immunization protocol (see Harlow and Lane, supra).Alternatively, a synthetic peptide, preferably near full length, derivedfrom the sequences disclosed herein and conjugated to a carrier proteincan be used an immunogen. Polyclonal sera are collected and titeredagainst the immunogen protein in an immunoassay, for example, a solidphase immunoassay with the immunogen immobilized on a solid support.Polyclonal antisera with a titer of 10⁴ or greater are selected andtested for their cross reactivity against C, C—C, and CXC chemokines,using a competitive binding immunoassay such as the one described inHarlow and Lane, supra, at pages 570-573. Preferably two chemokines areused in this determination in conjunction with either human CX3Ckine ormouse CX3Ckine.

In conjunction with a CX3Ckine, the monocyte chemotactic protein-1(MCP-1) and macrophage inflammatory protein-1α (Mip-1α) are used toidentify antibodies which are specifically bound by a CX3Ckine. Inconjunction with human CX3Ckine, the monocyte chemotactic protein-2(MCP-2) and Mip-1α are used to identify antibodies which arespecifically bound by a CX3Ckine. These chemokines can be produced asrecombinant proteins and isolated using standard molecular biology andprotein chemistry techniques as described herein.

Immunoassays in the competitive binding format can be used for thecrossreactivity determinations. For example, a protein of SEQ ID NO: 2.4, 6, or 8 can be immobilized to a solid support. Proteins added to theassay compete with the binding of the antisera to the immobilizedantigen. The ability of the above proteins to compete with the bindingof the antisera to the immobilized protein is compared to the protein ofSEQ ID NO: 2. 4, 6, or 8. The percent crossreactivity for the aboveproteins is calculated, using standard calculations. Those antisera withless than 10% crossreactivity with each of the proteins listed above areselected and pooled. The cross-reacting antibodies are then removed fromthe pooled antisera by immunoabsorbtion with the above-listed proteins.

The immunoabsorbed and pooled antisera are then used in a competitivebinding immunoassay as described above to compare a second protein tothe immunogen protein (e.g., the CX3Ckine chemokine motif of SEQ ID NO:2. 4, 6, or 8). In order to make this comparison, the two proteins areeach assayed at a wide range of concentrations and the amount of eachprotein required to inhibit 50% of the binding of the antisera to theimmobilized protein is determined. If the amount of the second proteinrequired is less than twice the amount of the protein of SEQ ID NO: 2that is required, then the second protein is said to specifically bindto an antibody generated to the immunogen.

It is understood that CX3Ckine proteins are a family of homologousproteins that comprise two or more genes. For a particular gene product,such as the human CX3Ckine protein, the term refers not only to theamino acid sequences disclosed herein, but also to other proteins thatare polymorphic, allelic, non-allelic, or species variants. It is alsounderstood that the term “human CX3Ckine” or “mouse CX3Ckine” includesnormatural mutations introduced by deliberate mutation usingconventional recombinant technology such as single site mutation, or byexcising short sections of DNA encoding CX3Ckine proteins, or bysubstituting new amino acids, or adding new amino acids. Such minoralterations must substantially maintain the immunoidentity of theoriginal molecule and/or its biological activity. Thus, thesealterations include proteins that are specifically immunoreactive with adesignated naturally occurring CX3Ckine protein, for example, the humanCX3Ckine protein shown in SEQ ID NO: 2 or 4. The biological propertiesof the altered proteins can be determined by expressing the protein inan appropriate cell line and measuring, e.g., a chemotactic effect.Particular protein modifications considered minor would includeconservative substitution of amino acids with similar chemicalproperties, as described above for the CX3Ckine family as a whole. Byaligning a protein optimally with the protein of SEQ ID NO: 2. 4, 6, or8, and by using the conventional immunoassays described herein todetermine immunoidentity, or by using lymphocyte chemotaxis assays, onecan determine the protein compositions of the invention.

IX. Functional Variants

The blocking of physiological response to CX3Ckines may result from theinhibition of binding of the protein to its receptor, e.g., throughcompetitive inhibition. Thus, in vitro assays of the present inventionwill often use isolated protein, membranes from cells expressing arecombinant membrane associated CX3Ckine, soluble fragments comprisingreceptor binding segments of these proteins, or fragments attached tosolid phase substrates. These assays will also allow for the diagnosticdetermination of the effects of either binding segment mutations andmodifications, or protein mutations and modifications, e.g., proteinanalogs. This invention also contemplates the use of competitive drugscreening assays, e.g., where neutralizing antibodies to antigen orreceptor fragments compete with a test compound for binding to theprotein. In this manner, the antibodies can be used to detect thepresence of a polypeptide which shares one or more antigenic bindingsites of the protein and can also be used to occupy binding sites on theprotein that might otherwise interact with a receptor.

“Derivatives” of CX3Ckine antigens include amino acid sequence mutants,glycosylation variants, and covalent or aggregate conjugates with otherchemical moieties. Covalent derivatives can be prepared by linkage offunctionalities to groups which are found in CX3Ckine amino acid sidechains or at the N- or C-termini, by means which are well known in theart. These derivatives can include, without limitation, aliphatic estersor amides of the carboxyl terminus, or of residues containing carboxylside chains, O-acyl derivatives of hydroxyl group-containing residues,and N-acyl derivatives of the amino terminal amino acid or amino-groupcontaining residues, e.g., lysine or arginine. Acyl groups are selectedfrom the group of alkyl-moieties including C3 to C18 normal alkyl,thereby forming alkanoyl aroyl species. See, e.g., Coligan, et al.(eds.) (1995 and periodic supplements) Current Protocols in ProteinScience, John Wiley and Sons, New York, N.Y. Covalent attachment tocarrier proteins may be important when immunogenic moieties are haptens.

In particular, glycosylation alterations are included, e.g., made bymodifying the glycosylation patterns of a polypeptide during itssynthesis and processing, or in further processing steps. Particularlypreferred means for accomplishing this are by exposing the polypeptideto glycosylating enzymes derived from cells which normally provide suchprocessing, e.g., mammalian glycosylation enzymes. Deglycosylationenzymes are also contemplated. Also embraced are versions of the sameprimary amino acid sequence which have other minor modifications,including phosphorylated amino acid residues, e.g., phosphotyrosine,phosphoserine, or phosphothreonine, or other moieties, including ribosylgroups or cross-linking reagents.

A major group of derivatives are covalent conjugates of the CX3Ckine orfragments thereof with other proteins or polypeptides. These derivativescan be synthesized in recombinant culture such as N- or C-terminalfusions or by the use of agents known in the art for their usefulness incross-linking proteins through reactive side groups. Preferred proteinderivatization sites with cross-linking agents are at free amino groups,carbohydrate moieties, and cysteine residues.

Fusion polypeptides between CX3Ckines and other homologous orheterologous proteins are also provided. Many growth factors andcytokines are homodimeric entities, and a repeat construct may havevarious advantages, including lessened susceptibility to proteolyticdegradation. Moreover, many receptors require dimerization to transducea signal, and various dimeric proteins or domain repeats can bedesirable. Heterologous polypeptides may be fusions between differentsurface markers, resulting in, e.g., a hybrid protein exhibitingreceptor binding specificity. Likewise, heterologous fusions may beconstructed which would exhibit a combination of properties oractivities of the derivative proteins. Typical examples are fusions of areporter polypeptide, e.g., luciferase, with a segment or domain of aprotein, e.g., a receptor-binding segment, so that the presence orlocation of the fused protein may be easily determined. See, e.g., Dull,et al., U.S. Pat. No. 4,859,609. Other gene fusion partners includebacterial β-galactosidase, trpE, Protein A, β-lactamase, alpha amylase,alcohol dehydrogenase, and yeast alpha mating factor. See, e.g., See,e.g., Dawson, et al. (1994) Science 266:776-779; and Godowski, et al.(1988) Science 241:812-816. In particular, fusion proteins with portionsfrom the related genes will be useful. Similar concepts of fusions withantigen binding sites are contemplated.

Such polypeptides may also have amino acid residues which have beenchemically modified by phosphorylation, sulfonation, biotinylation, orthe addition or removal of other moieties, particularly those which havemolecular shapes similar to phosphate groups. In some embodiments, themodifications will be useful labeling reagents, or serve as purificationtargets, e.g., affinity ligands.

This invention also contemplates the use of derivatives of CX3Ckinesother than variations in amino acid sequence or glycosylation. Suchderivatives may involve covalent or aggregative association withchemical moieties. These derivatives include: (1) salts, (2) side chainand terminal residue covalent modifications, and (3) adsorptioncomplexes, for example with cell membranes. Such covalent or aggregativederivatives are useful as immunogens, as reagents in immunoassays, or inpurification methods such as for affinity purification of ligands orother binding ligands. For example, a CX3Ckine antigen can beimmobilized by covalent bonding to a solid support such as cyanogenbromide-activated SEPHAROSE, by methods which are well known in the art,or adsorbed onto polyolefin surfaces, with or without glutaraldehydecross-linking, for use in the assay or purification of anti-CX3Ckineantibodies or its receptor. The CX3Ckines can also be labeled with adetectable group, e.g., radioiodinated by the chloramine T procedure,covalently bound to rare earth chelates, or conjugated to anotherfluorescent moiety for use in diagnostic assays. Purification ofCX3Ckines may be effected by immobilized antibodies or receptor.

Isolated CX3Ckine genes will allow transformation of cells lackingexpression of corresponding CX3Ckines, e.g., either species types orcells which lack corresponding proteins and exhibit negative backgroundactivity. Expression of transformed genes will allow isolation ofantigenically pure cell lines, with defined or single specie variants.This approach will allow for more sensitive detection and discriminationof the physiological effects of CX3Ckine receptor proteins. Subcellularfragments, e.g., cytoplasts or membrane fragments, can be isolated andused.

X. Uses

The present invention provides reagents which will find use indiagnostic applications as described elsewhere herein, e.g., in thegeneral description for developmental abnormalities, or below in thedescription of kits for diagnosis.

CX3Ckine nucleotides, e.g., human or mouse CX3Ckine DNA or RNA, may beused as a component in a forensic assay. For instance, the nucleotidesequences provided may be labeled using, e.g., ³²P or biotin and used toprobe standard restriction fragment polymorphism blots, providing ameasurable character to aid in distinguishing between individuals. Suchprobes may be used in well-known forensic techniques such as geneticfingerprinting. In addition, nucleotide probes made from CX3Ckinesequences may be used in in situ assays to detect chromosomalabnormalities. For instance, rearrangements in the mouse chromosomeencoding a CX3Ckine gene may be detected via well-known in situtechniques, using CX3Ckine probes in conjunction with other knownchromosome markers.

Antibodies and other binding agents directed towards CX3Ckine proteinsor nucleic acids may be used to purify the corresponding CX3Ckinemolecule. As described in the Examples below, antibody purification ofCX3Ckine components is both possible and practicable. Antibodies andother binding agents may also be used in a diagnostic fashion todetermine whether CX3Ckine components are present in a tissue sample orcell population using well-known techniques described herein. Theability to attach a binding agent to a CX3Ckine provides a means todiagnose disorders associated with CX3Ckine misregulation. Antibodiesand other CX3Ckine binding agents may also be useful as histologicalmarkers. As described in the examples below, CX3Ckine expression islimited to specific tissue types. By directing a probe, such as anantibody or nucleic acid to a CX3Ckine it is possible to use the probeto distinguish tissue and cell types in situ or in vitro.

This invention also provides reagents with significant therapeuticvalue. The CX3Ckines (naturally occurring or recombinant), fragmentsthereof, and antibodies thereto, along with compounds identified ashaving binding affinity to a CX3Ckine, are useful in the treatment ofconditions associated with abnormal physiology or development, includingabnormal proliferation, e.g., cancerous conditions, or degenerativeconditions. Abnormal proliferation, regeneration, degeneration, andatrophy may be modulated by appropriate therapeutic treatment using thecompositions provided herein. For example, a disease or disorderassociated with abnormal expression or abnormal signaling by a CX3Ckineis a target for an agonist or antagonist of the protein. The proteinslikely play a role in regulation or development of neuronal orhematopoietic cells, e.g., lymphoid cells, which affect immunologicalresponses.

Other abnormal developmental conditions are known in cell types shown topossess CX3Ckine mRNA by northern blot analysis. See Berkow (ed.) TheMerck Manual of Diagnosis and Therapy, Merck & Co., Rahway, N.J.; andThorn, et al. Harrison's Principles of Internal Medicine, McGraw-Hill,N.Y. Developmental or functional abnormalities, e.g., of the neuronal orimmune system, cause significant medical abnormalities and conditionswhich may be susceptible to prevention or treatment using compositionsprovided herein.

Certain chemokines have also been implicated in viral replicationmechanisms. See, e.g., Cohen (1996) Science 272:809-810; Feng, et al.(1996) Science 272:872-877; and Cocchi, et al. (1995) Science270:1811-1816. The CX3C chemokine may be useful in a similar context.Alternatively, the stalk structure may be very important in presentationof the ligand domain, and other chemokines may be advantageouslysubstituted for the chemokine domain in this molecule. Modification inthe “stalk” structure may affect many of the pharmacological propertiesof the CX3Ckine, including half-life and biological activity.

Recombinant CX3Ckine or CX3Ckine antibodies can be purified and thenadministered to a patient, e.g., in sterile form. These reagents can becombined for therapeutic use with additional active or inertingredients, e.g., in conventional pharmaceutically acceptable carriersor diluents, e.g., immunogenic adjuvants, along with physiologicallyinnocuous stabilizers and excipients. These combinations can be sterilefiltered and placed into dosage forms as by lyophilization in dosagevials or storage in stabilized aqueous preparations. This invention alsocontemplates use of antibodies or binding fragments thereof, includingforms which are not complement binding.

Drug screening using antibodies or receptor or fragments thereof canidentify compounds having binding affinity to CX3Ckines, includingisolation of associated components. Subsequent biological assays canthen be utilized to determine if the compound has intrinsic stimulatingactivity and is therefore a blocker or antagonist in that it blocks theactivity of the protein. Likewise, a compound having intrinsicstimulating activity can activate the receptor and is thus an agonist inthat it simulates the activity of a CX3Ckine. This invention furthercontemplates the therapeutic use of antibodies to CX3Ckines asantagonists. This approach should be particularly useful with otherCX3Ckine species variants.

The quantities of reagents necessary for effective therapy will dependupon many different factors, including means of administration, targetsite, physiological state of the patient, and other medicantsadministered. Thus, treatment dosages should be titrated to optimizesafety and efficacy. Typically, dosages used in vitro may provide usefulguidance in the amounts useful for in situ administration of thesereagents. Animal testing of effective doses for treatment of particulardisorders will provide further predictive indication of human dosage.Various considerations are described, e.g., in Gilman, et al. (eds.)(1990) Goodman and Gilman's: The Pharmacological Bases of Therapeutics(8th ed.) Pergamon Press; and (1990) Remington's Pharmaceutical Sciences(17th ed.) Mack Publishing Co., Easton, Pa. Methods for administrationare discussed therein and below, e.g., for oral, intravenous,intraperitoneal, or intramuscular administration, transdermal diffusion,and others. Pharmaceutically acceptable carriers will include water,saline, buffers, and other compounds described, e.g., in the MerckIndex, Merck & Co., Rahway, N.J. Dosage ranges would ordinarily beexpected to be in amounts lower than 1 mM concentrations, typically lessthan about 10 μM concentrations, usually less than about 100 nM,preferably less than about 10 μM (picomolar), and most preferably lessthan about 1 fM (femtomolar), with an appropriate carrier. Slow releaseformulations, or a slow release apparatus will often be utilized forcontinuous administration.

CX3Ckines, fragments thereof, and antibodies to it or its fragments,antagonists, and agonists, may be administered directly to the host tobe treated or, depending on the size of the compounds, it may bedesirable to conjugate them to carrier proteins such as ovalbumin orserum albumin prior to their administration. Therapeutic formulationsmay be administered in many conventional dosage formulations. While itis possible for the active ingredient to be administered alone, it ispreferable to present it as a pharmaceutical formulation. Formulationstypically comprise at least one active ingredient, as defined above,together with one or more acceptable carriers thereof. Each carriershould be both pharmaceutically and physiologically acceptable in thesense of being compatible with the other ingredients and not injuriousto the patient. Formulations include those suitable for oral, rectal,nasal, or parenteral (including subcutaneous, intramuscular, intravenousand intradermal) administration. The formulations may conveniently bepresented in unit dosage form and may be prepared by any methods wellknown in the art of pharmacy. See, e.g., Gilman, et al. (eds.) (1990)Goodman and Gilman's: The Pharmacological Bases of Therapeutics (8thed.) Pergamon Press; and (1990) Remington's Pharmaceutical Sciences(17th ed.) Mack Publishing Co., Easton, Pa.; Avis, et al. (eds.) (1993)Pharmaceutical Dosage Forms: Parenteral Medications Dekker, NY;Lieberman, et al. (eds.) (1990) Pharmaceutical Dosage Forms: TabletsDekker, NY; and Lieberman, et al. (eds.) (1990) Pharmaceutical DosageForms: Disperse Systems Dekker, NY. The therapy of this invention may becombined with or used in association with other therapeutic agents.

Both the naturally occurring and the recombinant forms of the CX3Ckinesof this invention are particularly useful in kits and assay methodswhich are capable of screening compounds for binding activity to theproteins. Several methods of automating assays have been developed inrecent years so as to permit screening of tens of thousands of compoundsin a short period. See, e.g., Fodor, et al. (1991) Science 251:767-773,and other descriptions of chemical diversity libraries, which describemeans for testing of binding affinity by a plurality of compounds. Thedevelopment of suitable assays can be greatly facilitated by theavailability of large amounts of purified, soluble CX3Ckine as providedby this invention.

For example, antagonists can normally be found once the protein has beenstructurally defined. Testing of potential protein analogs is nowpossible upon the development of highly automated assay methods using apurified receptor. In particular, new agonists and antagonists will bediscovered by using screening techniques described herein. Of particularimportance are compounds found to have a combined binding affinity formultiple CX3Ckine receptors, e.g., compounds which can serve asantagonists for species variants of a CX3Ckine.

This invention is particularly useful for screening compounds by usingrecombinant protein in a variety of drug screening techniques. Theadvantages of using a recombinant protein in screening for specificligands include: (a) improved renewable source of the CX3Ckine from aspecific source; (b) potentially greater number of ligands per cellgiving better signal to noise ratio in assays; and (c) species variantspecificity (theoretically giving greater biological and diseasespecificity).

One method of drug screening utilizes eukaryotic or prokaryotic hostcells which are stably transformed with recombinant DNA moleculesexpressing a CX3Ckine receptor. Cells may be isolated which express areceptor in isolation from any others. Such cells, either in viable orfixed form, can be used for standard ligand/receptor binding assays. Seealso, Parce, et al. (1989) Science 246:243-247; and Owicki, et al.(1990) Proc. Nat'l Acad. Sci. USA 87:4007-4011, which describe sensitivemethods to detect cellular responses. Competitive assays areparticularly useful, where the cells (source of CX3Ckine) are contactedand incubated with a labeled receptor or antibody having known bindingaffinity to the ligand, such as ¹²⁵I-antibody, and a test sample whosebinding affinity to the binding composition is being measured. The boundand free labeled binding compositions are then separated to assess thedegree of ligand binding. The amount of test compound bound is inverselyproportional to the amount of labeled receptor binding to the knownsource. Any one of numerous techniques can be used to separate boundfrom free ligand to assess the degree of ligand binding. This separationstep could typically involve a procedure such as adhesion to filtersfollowed by washing, adhesion to plastic followed by washing, orcentrifugation of the cell membranes. Viable cells could also be used toscreen for the effects of drugs on CX3Ckine mediated functions, e.g.,second messenger levels, i.e., Ca⁺⁺; cell proliferation; inositolphosphate pool changes; and others. Some detection methods allow forelimination of a separation step, e.g., a proximity sensitive detectionsystem. Calcium sensitive dyes will be useful for detecting Ca⁺⁺ levels,with a fluorimeter or a fluorescence cell sorting apparatus.

Another method utilizes membranes from transformed eukaryotic orprokaryotic host cells as the source of a CX3Ckine. These cells arestably transformed with DNA vectors directing the expression of aCX3Ckine, e.g., an engineered membrane bound form. Essentially, themembranes would be prepared from the cells and used in a receptor/ligandbinding assay such as the competitive assay set forth above.

Still another approach is to use solubilized, unpurified or solubilized,purified CX3Ckine from transformed eukaryotic or prokaryotic host cells.This allows for a “molecular” binding assay with the advantages ofincreased specificity, the ability to automate, and high drug testthroughput.

Another technique for drug screening involves an approach which provideshigh throughput screening for compounds having suitable binding affinityto a CX3Ckine antibody and is described in detail in Geysen, EuropeanPatent Application 84/03564, published on Sep. 13, 1984. First, largenumbers of different small peptide test compounds are synthesized on asolid substrate, e.g., plastic pins or some other appropriate surface,see Fodor, et al., supra. Then all the pins are reacted withsolubilized, unpurified or solubilized, purified CX3Ckine antibody, andwashed. The next step involves detecting bound CX3Ckine antibody.

Rational drug design may also be based upon structural studies of themolecular shapes of the CX3Ckine and other effectors or analogs. See,e.g., Methods in Enzymology vols 202 and 203. Effectors may be otherproteins which mediate other functions in response to ligand binding, orother proteins which normally interact with the receptor. One means fordetermining which sites interact with specific other proteins is aphysical structure determination, e.g., x-ray crystallography or 2dimensional NMR techniques. These will provide guidance as to whichamino acid residues form molecular contact regions. For a detaileddescription of protein structural determination, see, e.g., Blundell andJohnson (1976) Protein Crystallography Academic Press, NY.

A purified CX3Ckine can be coated directly onto plates for use in theaforementioned drug screening techniques. However, non-neutralizingantibodies to these ligands can be used as capture antibodies toimmobilize the respective ligand on the solid phase.

XI. Kits

This invention also contemplates use of CX3Ckine proteins, fragmentsthereof, peptides, and their fusion products in a variety of diagnostickits and methods for detecting the presence of CX3Ckine or a CX3Ckinereceptor. Typically the kit will have a compartment containing either adefined CX3Ckine peptide or gene segment or a reagent which recognizesone or the other, e.g., receptor fragments or antibodies.

A kit for determining the binding affinity of a test compound to aCX3Ckine would typically comprise a test compound; a labeled compound,e.g., a receptor or antibody having known binding affinity for theCX3Ckine; a source of CX3Ckine (naturally occurring or recombinant); anda means for separating bound from free labeled compound, such as a solidphase for immobilizing the CX3Ckine. Once compounds are screened, thosehaving suitable binding affinity to the CX3Ckine can be evaluated insuitable biological assays, as are well known in the art, to determinewhether they act as agonists or antagonists to the receptor. Theavailability of recombinant CX3Ckine polypeptides also provide welldefined standards for calibrating such assays.

A preferred kit for determining the concentration of, for example, aCX3Ckine in a sample would typically comprise a labeled compound, e.g.,receptor or antibody, having known binding affinity for the CX3Ckine, asource of CX3Ckine (naturally occurring or recombinant), and a means forseparating the bound from free labeled compound, for example, a solidphase for immobilizing the CX3Ckine. Compartments containing reagents,and instructions, will normally be provided.

Antibodies, including antigen binding fragments, specific for theCX3Ckine or ligand fragments are useful in diagnostic applications todetect the presence of elevated levels of CX3Ckine and/or its fragments.Such may allow diagnosis of the amounts of differently processed formsof the CX3Ckine, e.g., successively degraded stalk structure. Suchdiagnostic assays can employ lysates, live cells, fixed cells,immunofluorescence, cell cultures, body fluids, and further can involvethe detection of antigens related to the ligand in serum, or the like.Diagnostic assays may be homogeneous (without a separation step betweenfree reagent and antigen-CX3Ckine complex) or heterogeneous (with aseparation step). Various commercial assays exist, such asradioimmunoassay (RIA), enzyme-linked immunosorbentassay (ELISA), enzymeimmunoassay (EIA), enzyme-multiplied immunoassay technique (EMIT),substrate-labeled fluorescent immunoassay (SLFIA), and the like. Forexample, unlabeled antibodies can be employed by using a second antibodywhich is labeled and which recognizes the antibody to a CX3Ckine or to aparticular fragment thereof. Similar assays have also been extensivelydiscussed in the literature. See, e.g., Harlow and Lane (1988)Antibodies: A Laboratory Manual, CSH Press, NY; Chan (ed.) (1987)Immunoassay: A Practical Guide Academic Press, Orlando, Fla.; Price andNewman (eds.) (1991) Principles and Practice of Immunoassay StocktonPress, NY; and Ngo (ed.) (1988) Nonisotopic Immunoassay Plenum Press,NY.

Anti-idiotypic antibodies may have similar use to diagnose presence ofantibodies against a CX3Ckine, as such may be diagnostic of variousabnormal states. For example, overproduction of CX3Ckine may result inproduction of various immunological or other medical reactions which maybe diagnostic of abnormal physiological states, e.g., in cell growth,activation, or differentiation.

Frequently, the reagents for diagnostic assays are supplied in kits, soas to optimize the sensitivity of the assay. For the subject invention,depending upon the nature of the assay, the protocol, and the label,either labeled or unlabeled antibody or receptor, or labeled CX3Ckine isprovided. This is usually in conjunction with other additives, such asbuffers, stabilizers, materials necessary for signal production such assubstrates for enzymes, and the like. Preferably, the kit will alsocontain instructions for proper use and disposal of the contents afteruse. Typically the kit has compartments for each useful reagent.Desirably, the reagents are provided as a dry lyophilized powder, wherethe reagents may be reconstituted in an aqueous medium providingappropriate concentrations of reagents for performing the assay.

Many of the aforementioned constituents of the drug screening and thediagnostic assays may be used without modification, or may be modifiedin a variety of ways. For example, labeling may be achieved bycovalently or non-covalently joining a moiety which directly orindirectly provides a detectable signal. In any of these assays, theprotein, test compound, CX3Ckine, or antibodies thereto can be labeledeither directly or indirectly. Possibilities for direct labeling includelabel groups: radiolabels such as ¹²⁵I, enzymes (U.S. Pat. No.3,645,090) such as peroxidase and alkaline phosphatase, and fluorescentlabels (U.S. Pat. No. 3,940,475) capable of monitoring the change influorescence intensity, wavelength shift, or fluorescence polarization.Possibilities for indirect labeling include biotinylation of oneconstituent followed by binding to avidin coupled to one of the abovelabel groups.

There are also numerous methods of separating the bound from the freeligand, or alternatively the bound from the free test compound. TheCX3Ckine can be immobilized on various matrices followed by washing.Suitable matrices include plastic such as an ELISA plate, filters, andbeads. Methods of immobilizing the CX3Ckine to a matrix include, withoutlimitation, direct adhesion to plastic, use of a capture antibody,chemical coupling, and biotin-avidin. The last step in this approachinvolves the precipitation of ligand/receptor or ligand/antibody complexby any of several methods including those utilizing, e.g., an organicsolvent such as polyethylene glycol or a salt such as ammonium sulfate.Other suitable separation techniques include, without limitation, thefluorescein antibody magnetizable particle method described in Rattle,et al. (1984) Clin. Chem. 30:1457-1461, and the double antibody magneticparticle separation as described in U.S. Pat. No. 4,659,678.

Methods for linking proteins or their fragments to the various labelshave been extensively reported in the literature and do not requiredetailed discussion here. Many of the techniques involve the use ofactivated carboxyl groups either through the use of carbodiimide oractive esters to form peptide bonds, the formation of thioethers byreaction of a mercapto group with an activated halogen such aschloroacetyl, or an activated olefin such as maleimide, for linkage, orthe like. Fusion proteins will also find use in these applications.

Another diagnostic aspect of this invention involves use ofoligonucleotide or polynucleotide sequences taken from the sequence of aCX3Ckine. These sequences can be used as probes for detecting levels ofthe CX3Ckine message in samples from natural sources, or patientssuspected of having an abnormal condition, e.g., cancer or developmentalproblem. The preparation of both RNA and DNA nucleotide sequences, thelabeling of the sequences, and the preferred size of the sequences hasreceived ample description and discussion in the literature. Normally anoligonucleotide probe should have at least about 14 nucleotides, usuallyat least about 18 nucleotides, and the polynucleotide probes may be upto several kilobases. Various labels may be employed, most commonlyradionuclides, particularly ³²P. However, other techniques may also beemployed, such as using biotin modified nucleotides for introductioninto a polynucleotide. The biotin then serves as the site for binding toavidin or antibodies, which may be labeled with a wide variety oflabels, such as radionuclides, fluorophores, enzymes, or the like.Alternatively, antibodies may be employed which can recognize specificduplexes, including DNA duplexes, RNA duplexes, DNA-RNA hybrid duplexes,or DNA-protein duplexes. The antibodies in turn may be labeled and theassay carried out where the duplex is bound to a surface, so that uponthe formation of duplex on the surface, the presence of antibody boundto the duplex can be detected. The use of probes to the novel anti-senseRNA may be carried out using many conventional techniques such asnucleic acid hybridization, plus and minus screening, recombinationalprobing, hybrid released translation (HRT), and hybrid arrestedtranslation (HART). This also includes amplification techniques such aspolymerase chain reaction (PCR).

Diagnostic kits which also test for the qualitative or quantitativepresence of other markers are also contemplated. Diagnosis or prognosismay depend on the combination of multiple indications used as markers.Thus, kits may test for combinations of markers. See, e.g., Viallet, etal. (1989) Progress in Growth Factor Res. 1:89-97.

XII. Receptor Isolation

Having isolated a binding partner of a specific interaction, methodsexist for isolating the counter-partner. See, Gearing, et al. (1989)EMBO J. 8:3667-3676. For example, means to label a CX3Ckine withoutinterfering with the binding to its receptor can be determined. Forexample, an affinity label or epitope tag can be fused to either theamino- or carboxyl-terminus of the ligand. An expression library can bescreened for specific binding of the CX3Ckine, e.g., by cell sorting, orother screening to detect subpopulations which express such a bindingcomponent. See, e.g., Ho, et al. (1993) Proc. Nat'l Acad. Sci. USA90:11267-11271. Alternatively, a panning method may be used. See, e.g.,Seed and Aruffo (1987) Proc. Nat'l Acad. Sci. USA 84:3365-3369. Atwo-hybrid selection system may also be applied making appropriateconstructs with the available BAS-1 sequences. See, e.g., Fields andSong (1989) Nature 340:245-246.

Protein cross-linking techniques with label can be applied to isolatebinding partners of a CX3Ckine. This would allow identification ofproteins which specifically interact with a CX3Ckine, e.g., in aligand-receptor like manner. Typically, the chemokine family binds toreceptors of the seven transmembrane receptor family, and the receptorfor the CX3Ckine is likely to exhibit a similar structure. Thus, it islikely that the receptor will be found by expression in a system whichis capable of expressing such a membrane protein in a form capable ofexhibiting ligand binding capability.

The broad scope of this invention is best understood with reference tothe following examples, which are not intended to limit the invention tospecific embodiments.

Examples I. General Methods

Many of the standard methods below are described or referenced, e.g., inManiatis, et al. (1982) Molecular Cloning, A Laboratory Manual ColdSpring Harbor Laboratory, Cold Spring Harbor Press, NY; Sambrook, et al.(1989) Molecular Cloning: A Laboratory Manual (2d ed.) Vols. 1-3, CSHPress, NY; Ausubel, et al., Biology Greene Publishing Associates,Brooklyn, N.Y.; or Ausubel, et al. (1987 and Supplements) CurrentProtocols in Molecular Biology Wiley/Greene, NY; Innis, et al. (eds.)(1990) PCR Protocols: A Guide to Methods and Applications AcademicPress, NY. Methods for protein purification include such methods asammonium sulfate precipitation, column chromatography, electrophoresis,centrifugation, crystallization, and others. See, e.g., Ausubel, et al.(1987 and periodic supplements); Deutscher (1990) “Guide to ProteinPurification,” Methods in Enzymology vol. 182, and other volumes in thisseries; and manufacturer's literature on use of protein purificationproducts, e.g., Pharmacia, Piscataway, N.J., or Bio-Rad, Richmond,Calif. Combination with recombinant techniques allow fusion toappropriate segments (epitope tags), e.g., to a FLAG sequence or anequivalent which can be fused, e.g., via a protease-removable sequence.See, e.g., Hochuli (1989) Chemische Industrie 12:69-70; Hochuli (1990)“Purification of Recombinant Proteins with Metal Chelate Absorbent” inSetlow (ed.) Genetic Engineering, Principle and Methods 12:87-98, PlenumPress, NY; Crowe, et al. (1992) QIAexpress: The High Level Expression &Protein Purification System QIAGEN, Inc., Chatsworth, Calif.; andColigan, et al. (eds.) (1995 and periodic supplements) Current Protocolsin Protein Science, John Wiley and Sons, New York, N.Y.

Standard immunological techniques are described, e.g., in Coligan (1991)Current Protocols in Immunology Wiley/Greene, NY; and Methods inEnzymology volumes. 70, 73, 74, 84, 92, 93, 108, 116, 121, 132, 150,162, and 163. Assays for neural cell biological activities aredescribed, e.g., in Wouterlood (ed. 1995) Neuroscience Protocols modules10, Elsevier; Methods in Neurosciences Academic Press; and NeuromethodsHumana Press, Totowa, N.J. Methodology of developmental systems isdescribed, e.g., in Meisami (ed.) Handbook of Human Growth andDevelopmental Biology CRC Press; and Chrispeels (ed.) MolecularTechniques and Approaches in Developmental Biology Interscience.

FACS analyses are described in Melamed, et al. (1990) Flow Cytometry andSorting Wiley-Liss, Inc., New York, N.Y.; Shapiro (1988) Practical FlowCytometry Liss, New York, N.Y.; and Robinson, et al. (1993) Handbook ofFlow Cytometry Methods Wiley-Liss, New York, N.Y.

II. Isolation of Human CX3Ckine Clone

A clone encoding the human CX3Ckine is isolated from a natural source bymany different possible methods. Given the sequences provided herein,PCR primers or hybridization probes are selected and/or constructed toisolate either genomic DNA segments or cDNA reverse transcripts.Appropriate cell sources include human tissues, e.g., brain libraries.Tissue distribution below also suggests source tissues. Genetic andpolymorphic or allelic variants are isolated by screening a populationof individuals.

PCR based detection is performed by standard methods, preferably usingprimers from opposite ends of the coding sequence, but flanking segmentsmight be selected for specific purposes.

Alternatively, hybridization probes are selected. Particular AT or GCcontents of probes are selected depending upon the expected homology andmismatching expected. Appropriate stringency conditions are selected tobalance an appropriate positive signal to background ratio. Successivewashing steps are used to collect clones of greater homology.

Further clones are isolated using an antibody based selection procedure.Standard expression cloning methods are applied including, e.g., FACSstaining of membrane associated expression product. The antibodies areused to identify clones producing a recognized protein. Alternatively,antibodies are used to purify a CX3C chemokine, with protein sequencingand standard means to isolate a gene encoding that protein.

Genomic sequence based methods will also allow for identification ofsequences naturally available, or otherwise, which exhibit homology tothe provided sequences. Similar procedures will allow isolation of otherprimate genes.

III. Isolation of Rodent CX3Ckine Clone

Similar methods are used as above to isolate an appropriate mouse CX3Cchemokine gene. Similar source materials as indicated above are used toisolate natural genes, including genetic, polymorphic, allelic, orstrain variants. Species variants are also isolated using similarmethods, e.g., from rats, moles, muskrats, copybaras, etc.

IV. Isolation of an avian CX3Ckine clone

An appropriate avian source is selected as above. Similar methods areutilized to isolate a species variant, though the level of similaritywill typically be lower for avian CX3C chemokine as compared to a humanto mouse sequence.

V. Expression; Purification; Characterization

With an appropriate clone from above, the coding sequence is insertedinto an appropriate expression vector. This may be in a vectorspecifically selected for a prokaryote, yeast, insect, or highervertebrate, e.g., mammalian expression system. Standard methods areapplied to produce the gene product, preferably as a soluble secretedmolecule, but will, in certain instances, also be made as anintracellular protein. Intracellular proteins typically require celllysis to recover the protein, and insoluble inclusion bodies are acommon starting material for further purification.

With a clone encoding a vertebrate CX3C chemokine, recombinantproduction means are used, although natural forms may be purified fromappropriate sources. The protein product is purified by standard methodsof protein purification, in certain cases, e.g., coupled withimmunoaffinity methods. Immunoaffinity methods are used either as apurification step, as described above, or as a detection assay todetermine the separation properties of the protein.

Preferably, the protein is secreted into the medium, and the solubleproduct is purified from the medium in a soluble form. Alternatively, asdescribed above, inclusion bodies from prokaryotic expression systemsare a useful source of material. Typically, the insoluble protein issolubilized from the inclusion bodies and refolded using standardmethods. Purification methods are developed as described above.

In certain embodiments, the protein is made in a eukaryotic cell whichglycosylates the protein normally. The purification methods may beaffected thereby, as may biological activities. The intact protein canbe processed to release the chemokine domain, probably due to a proteasecleavage event somewhere in the glycosylated stalk region close to thechemokine/stalk boundary. While recombinant protein appears to beprocessed, the physiological processes which normally do such in nativecells remain to be determined.

The product of the purification method described above is characterizedto determine many structural features. Standard physical methods areapplied, e.g., amino acid analysis and protein sequencing. The resultingprotein is subjected to CD spectroscopy and other spectroscopic methods,e.g., NMR, ESR, mass spectroscopy, etc. The product is characterized todetermine its molecular form and size, e.g., using gel chromatographyand similar techniques. Understanding of the chromatographic propertieswill lead to more gentle or efficient purification methods.

CX3C chemokine protein biochemistry was assessed in mammalian expressionsystems. Human embryonic kidney 293 cells (HEK 293) transfected with amammalian expression construct encoding full-length CX3C chemokine weremetabolically labeled with ³⁵S cysteine and methionine. CX3C chemokinewas produced as a protein of Mr ˜95 kDa; control transfectedsupernatants contained no such species. Neuraminidase and gycosidasesreduced the Mr of CX3C chemokine from ˜95 kDa to ˜45 kDa, suggestingthat the recombinant form is glycosylated substantially. Thus CX3Cchemokine cDNA, encoding a predicted membrane-bound protein, encodes aglycoprotein which is released from cells by an undefined mechanism.

Prediction of glycosylation sites may be made, e.g., as reported inHansen, et al. (1995) Biochem. J. 308:801-813.

VI. Preparation of Antibodies Against Vertebrate CX3Ckine

With protein produce, as above, animals are immunized to produceantibodies. Polyclonal antiserum is raised using non-purified antigen,though the resulting serum will exhibit higher background levels.Preferably, the antigen is purified using standard protein purificationtechniques, including, e.g., affinity chromatography using polyclonalserum indicated above. Presence of specific antibodies is detected usingdefined synthetic peptide fragments. Preferred fragments include thechemokine domain.

Polyclonal serum is raised against a purified antigen, purified asindicated above, or using synthetic peptides. A series of overlappingsynthetic peptides which encompass all of the full length sequence, ifpresented to an animal, will produce serum recognizing most linearepitopes on the protein. Such an antiserum is used to affinity purifyprotein, which is, in turn, used to introduce intact full length proteininto another animal to produce another antiserum preparation.

Similar techniques are used to generate induce monoclonal antibodies toeither unpurified antigen, or, preferably, purified antigen.

VII. Cellular and Tissue Distribution

Distribution of the protein or gene products are determined, e.g., usingimmunohistochemistry with an antibody reagent, as produced above, or byscreening for nucleic acids encoding the chemokine. Either hybridizationor PCR methods are used to detect DNA, cDNA, or message content.Histochemistry allows determination of the specific cell types within atissue which express higher or lower levels of message or DNA. Antibodytechniques are useful to quantitate protein in a biological sample,including a liquid or tissue sample. Immunoassays are developed toquantitate protein.

Hybridization techniques were applied to the tissue types in Table 3with positive or negative results, as indicated. The commercial tissueblots may have cellular contamination from resident cells, e.g., fromblood or other cells which populate the tissue. The large and smalltranscripts correspond to sizes about 4 kb and less than about 2 kb,respectively.

TABLE 3 Tissue and cell distribution of human CX3Ckine gene. Commercialtissue library: cell type large small spleen − − thymus + − prostate + +testis + − ovary + − small intestine + + colon + + peripheral blood − −

Further analysis of tissue distribution indicates abundance of humanmessage: heart +++; brain +++; placenta −; lung ++; liver −; muscle +;kidney −; pancreas +; spleen −; thymus +; prostate ++; testis +; ovary+; small intestine ++; colon ++; peripheral blood −; HL60 promyelocyticleukemia line −; HeLa cell S3−; K562 chronic myelogenous leukemia line−; Molt4 lymphoblastic leukemia line −; Burkitts lymphoma RAJI line −;SW480 colorectal adenocarcinoma line +; A549 lung carcinoma line −; andG361 melanoma line −.

“Reverse northerns” are blots from cDNA libraries with the insertsremoved, and the size determinations are based upon the size of insertsin the cDNA library, and reflect the lengths found in the cDNA libraryinserts, which may be less than full length where the reversetranscription was not full length. As such, size determinations thereare not reflective of the natural sizes. The results of these are: PBMC(peripheral blood mononuclear cells) +; PBMC (activated using T cellstimulation conditions, with anti-CD3 and PMA) −; Mot72 (resting Th0clone) +; Mot 72 (activated with anti-CD28 and anti-CD3) −; Mot72 α(activated with anti-peptide, anergic clone) −; Mot81 (resting Th0clone) −; Mot81 (activated with anti-CD28 and anti-CD3) −; HY06 (restingTh1 clone) −; HY06 (activated with anti-CD28 and anti-CD3) −; HY06α(activated with anti-peptide, anergic clone) −; HY935 (resting Th2clone) −; HY935 (activated with anti-CD28 and anti-CD3) +; BC pool ofEBV transformed lines +; resting splenocytes +; splenocytes +(activatedusing B cell stimulating conditions, with anti-CD40 and IL-4) −; NK cellpool −; NK pool (activated 6 h with PMA and ionomycin) +; NKA6 NK cellclone −; NKB1 NK cell clone −; NK non-cytotoxic cell clone +; and NKclone stimulated to be cytotoxic Other cells and tissues: CHO cells +;Jurkat cells (DNAX) +; Jurkat cells (another source) +; normal T cellpool +; TCT pool (transformed T cells) −; fetal kidney −; fetal lung −;fetal liver −; fetal heart −; fetal brain +; fetal gall bladder +; fetalsmall intestine +; fetal adipose +; fetal ovary −; fetal uterus +; adultplacenta −; fetal testis +; fetal spleen +; and fetal brain +.Additional cells provided: U937 (resting monocyte cell line) +; C—(elutriated monocyte activated with LPS, IFN-γ, and anti-IL-10) +;C+(elutriated monocytes activated with LPS, IFN-γ, and IL-10) +; M1(elutriated monocytes activated with LPS1 h)+; M6 (elutriated monocytesactivated with LPS 6 h) +; 30% DC (resting 30% CD1a+ dendritic cells,proliferated in TNF-α and GM-CSF)+; 70% DC (resting 70% CD1a+ dendriticcells, proliferated in TNF-α and GM-CSF) +; D1 (dendritic cellsstimulated 1 h in PMA and ionomycin)−; D6 (dendritic cells stimulated 6h in PMA and ionomycin) −; D5 DC (resting dendritic cells cultured 5 din GM-CSF and IL-4) +; DC (dendritic cells cultured in GM-CSF and IL-4,LPS activated) +; DC (GM-CSF activated, like D5 cells) +; DC mix(dendritic cells stimulated with a mixture of cytokines) +; CD1a+(99%pure CD1a+ dendritic cells, enriched from 70% DC) +; CD14+ (CD14+fraction sorted from 70% DC, monocyte-like morphology) −; CD1Aa+ (95%CD1a+ and CD86+ sorted from 70% DC) −; TF1 (hematopoietic precursorline) +; Jurkat (T cell line)+; MRC5 (lung fibroblast sarcoma cell line)+; JY (B cell line) +; U937 (pre-monocytic cell line) +.

Since the endothelium is a major site of chemokine action, a northernblot was performed to ascertain if CX3Ckine was expressed in thistissue. Human CX3Ckine was also shown to be expressed on human activatedprimary endothelial cells by both mRNA and protein expression. Thissuggests that CX3Ckine may be involved in leukocyte trafficking invarious organs.

In summary, human CX3Ckine mRNA is found in monocytes, dendritic cells,T cells and B cells, e.g., found in certain immune cells.

VIII. Microchemotaxis Assays

The pro-migratory activities of CX3C chemokine have been assessed inmicrochemotaxis assays. See, e.g., Bacon, et al. (1988) Br. J.Pharmacol. 95:966-974. CX3C chemokine appears to be a potent attractantof peripheral blood monocytes and T cells. Pro-migratory activity forblood neutrophils has been difficult to demonstrate.

IX. Chromosomal Mapping

The CX3C chemokine gene has been mapped to human chromosome 16. A BIOSLaboratories (New Haven, Conn.) mouse somatic cell hybrid panel wascombined with PCR. These mapping studies also indicate the possibilityof a pseudogene or related gene on human chromosome 14. Sequencing ofgenomic DNA fragments suggests CX3C chemokine gene has an intron whichbegins near or in the codon encoding Ile 64. Other intron/exonboundaries have yet to be mapped. This location is distinct from thechromosomal mapping locations of the other C, CC, or CXC chemokinefamilies, consistent with the CX3Ckine being a separate gene familywithin the chemokines.

X. Biological Activities, Direct and Indirect

The 293 human embryonic kidney cell line (HEK 293) was transfected witheither the membrane bound form of human CX3Ckine (293-CX3Ckine), thechemokine domain plus the “stalk” region, or a control vector without aninsert. The transfected cells were subsequently cultured with eithermonocytes, T cells, or peripheral mononuclear (PMN) cells to assayrelative adherence of these cells to CX3Ckine. Specifically, 5×10⁴ cellsper well of HEK 293 transfected cells were seeded in a 96 well plate.2×10⁵ monocytes, T cells, or PMNs, metabolically labeled with³⁵S-methionine and cysteine (Amersham, Arlington Heights, Ill.), wereadded to each well. The plate was then incubated at 37° C. for variustime points. The wells were washed 2 times RPMI supplemented with 1%FCS. Plates were then read in a Millipore Cytofluor at 485/530 nm.

In all cases, adherence to HEK 293 cells transfected with the membranebound form of CX3Ckine was significantly enhanced when compared to thetruncated CX3Ckine or mock transfected cells. Interestingly, only themembrane bound form possessed this proadhesive activity, leading to theconclusion that CX3Ckine, in its membrane bound form, may serve as aregulator of circulating leukocytes.

In another experiment, the recombinant soluble form of the chemokinedomain of CX3Ckine (rCx3C) was added to HEK 293-CX3C cells and monocytesat a concentration of 1 μM per well, and assayed as described above.rCX3C was able to antagonize adhesion of monocytes to HEK 293-CX3Ccells. A similar experiment was performed to investigate the effect on Tcell adherence. Comparable results were obtained. Thus rCX3C mayfunction as a negative regulator of circulating leukocytes.

A comparison of three different forms of human CX3Ckine was performed toanalyze variations in chemoattractant activity that may be due to thestructure of CX3Ckine. CX3C 1.7 (chemokine domain plus the entire stalkregion), CX3C 0.7 (chemokine domain plus one-half stalk region), andCX3C CK (chemokine domain only) were subjected to the chemotaxicityassay described above, their ability to attract T cells was analyzed.CX3C 1.7 displayed a slightly better dose dependent ability to attract Tcells relative to the other forms of CX3Ckine.

A robust and sensitive assay is selected as described above, e.g., onimmune cells, neuronal cells, or stem cells. Chemokine is added to theassay in increasing doses to see if a dose response is detected. Forexample, in a proliferation assay, cells are plated out in plates.Appropriate culture medium is provided, and chemokine is added to thecells in varying amounts. Growth is monitored over a period of timewhich will detect either a direct effect on the cells, or an indirecteffect of the chemokine.

Alternatively, an activation assay or attraction assay is used. Anappropriate cell type is selected, e.g, hematopoietic cells, myeloid(macrophages, neutrophils, polymorphonuclear cells, etc.) or lymphoid (Tcell, B cell, or NK cells), neural cells (neurons, neuroglia,oligodendrocytes, astrocytes, etc.), or stem cells, e.g., progenitorcells which differentiate to other cell types, e.g., gut crypt cells andundifferentiated cell types.

Other assays will be those which have been demonstrated with otherchemokines. See, e.g., Schall and Bacon (1994) Current Opinion inImmunology 6:865-873; and Bacon and Schall (1996) Int. Arch. Allergy &Immunol. 109:97-109. Effects of truncated stalk structures will besimilarly evaluated.

XI. Structure Activity Relationship

Information on the criticality of particular residues is determinedusing standard procedures and analysis. Standard mutagenesis analysis isperformed, e.g., by generating many different variants at determinedpositions, e.g., at the positions identified above, and evaluatingbiological activities of the variants. This may be performed to theextent of determining positions which modify activity, or to focus onspecific positions to determine the residues which can be substituted toeither retain, block, or modulate biological activity.

Alternatively, analysis of natural variants can indicate what positionstolerate natural mutations. This may result from populational analysisof variation among individuals, or across strains or species. Samplesfrom selected individuals are analysed, e.g., by PCR analysis andsequencing. This allows evaluation of population polymorphisms.Particularly, as described above, many of the biological activities ofthe chemokine domain attached to different portions or extents of thestalk structure may result.

XII. Screening for Agonists/Antagonists

Agonists or antagonists are screened for ability to induce or blockbiological activity. The candidate compounds, e.g, sequence variants ofnatural CX3Ckines, are assayed for their biological activities.Alternatively, compounds are screened, alone or in combinations, todetermine effects on biological activity.

XIII. Isolation of a Receptor for CX3C Chemokine

Based on the proadherent properties of CX3Ckine, 7 transmembraneG-protein receptor was found to be expressed by monocytes and T cells.It was also discovered that the chemokine domain is the only region ofCX3Ckine that can engage the receptor. Binding assays with knownchemokine receptor revealed that CX3Ckine does not engage chemokinereceptors CCR 1 through 5, CXCR 1 and 2, or the Duffy antigen receptor.CX3Ckine can, however, bind to a virally encoded chemokine receptor,CMV-US28.

Alternatively, CX3C chemokine can be used as a specific binding reagentto identify its binding partner, by taking advantage of its specificityof binding, much like an antibody would be used. A binding reagent iseither labeled as described above, e.g., fluorescence or otherwise, orimmobilized to a substrate for panning methods. The typical chemokinereceptor is a seven transmembrane receptor.

The purified protein is also be used to identify other binding partnersof CX3Ckine as described, e.g., in Fields and Song (1989) Nature340:245-246.

The binding composition, e.g., chemokine, is used to screen anexpression library made from a cell line which expresses a bindingpartner, i.e. receptor. Standard staining techniques are used to detector sort intracellular or surface expressed receptor, or surfaceexpressing transformed cells are screened by panning. Screening ofintracellular expression is performed by various staining orimmunofluorescence procedures. See also McMahan, et al. (1991) EMBO J.10:2821-2832.

For example, on day 0, precoat 2-chamber permanox slides with 1 ml perchamber of fibronectin, 10 ng/ml in PBS, for 30 min at room temperature.Rinse once with PBS. Then plate COS cells at 2−3×10⁵ cells per chamberin 1.5 ml of growth media. Incubate overnight at 37 C.

On day 1 for each sample, prepare 0.5 ml of a solution of 66 μg/mlDEAE-dextran, 66 μM chloroquine, and 4 μg DNA in serum free DME. Foreach set, a positive control is prepared, e.g., of human CX3C chemokinecDNA at 1 and 1/200 dilution, and a negative mock. Rinse cells withserum free DME. Add the DNA solution and incubate 5 hr at 37 C. Removethe medium and add 0.5 ml 10% DMSO in DME for 2.5 min. Remove and washonce with DME. Add 1.5 ml growth medium and incubate overnight.

On day 2, change the medium. On days 3 or 4, the cells are fixed andstained. Rinse the cells twice with Hank's Buffered Saline Solution(HBSS) and fix in 4% paraformaldehyde (PFA)/glucose for 5 min. Wash 3×with HBSS. The slides may be stored at −80 C after all liquid isremoved. For each chamber, 0.5 ml incubations are performed as follows.Add HBSS/saponin (0.1%) with 32 μl/ml of 1 M NaN₃ for 20 min. Cells arethen washed with HBSS/saponin 1×. Add chemokine or chemokine/antibodycomplex to cells and incubate for 30 min. Wash cells twice withHBSS/saponin. If appropriate, add first antibody for 30 min. Add secondantibody, e.g., Vector anti-mouse antibody, at 1/200 dilution, andincubate for 30 min. Prepare ELISA solution, e.g., Vector Elite ABChorseradish peroxidase solution, and preincubate for 30 min. Use, e.g.,1 drop of solution A (avidin) and 1 drop solution B (biotin) per 2.5 mlHBSS/saponin. Wash cells twice with HBSS/saponin. Add ABC HRP solutionand incubate for 30 min. Wash cells twice with HBSS, second wash for 2min, which closes cells. Then add Vector diaminobenzoic acid (DAB) for 5to 10 min. Use 2 drops of buffer plus 4 drops DAB plus 2 drops of H₂O₂per 5 ml of glass distilled water. Carefully remove chamber and rinseslide in water. Air dry for a few minutes, then add 1 drop of CrystalMount and a cover slip. Bake for 5 min at 85-90 C.

Evaluate positive staining of pools and pregressively subclone toisolation of single genes responsible for the binding.

Alternatively, chemokine reagents are used to affinity purify or sortout cells expressing a receptor. See, e.g., Sambrook, et al. or Ausubel,et al.

Another strategy is to screen for a membrane bound receptor by panning.The receptor cDNA is constructed as described above. The ligand can beimmobilized and used to immobilize expressing cells. Immobilization maybe achieved by use of appropriate antibodies which recognize, e.g., aFLAG sequence of a chemokine fusion construct, or by use of antibodiesraised against the first antibodies. Recursive cycles of selection andamplification lead to enrichment of appropriate clones and eventualisolation of receptor expressing clones. Phage expression libraries canbe screened by chemokine. Appropriate label techniques, e.g., anti-FLAGantibodies, will allow specific labeling of appropriate clones.

SEQUENCES

SEQ ID NO: 1 is human CX3Ckine nucleotide sequence.

SEQ ID NO: 2 is human CX3Ckine amino acid sequence.

SEQ ID NO: 3 is more complete human CX3Ckine nucleotide sequence.

SEQ ID NO: 4 is more complete human CX3Ckine amino acid sequence.

SEQ ID NO: 5 is partial mouse CX3Ckine nucleotide sequence.

SEQ ID NO: 6 is partial mouse CX3Ckine amino acid sequence.

SEQ ID NO: 7 is complete mouse CX3Ckine nucleotide sequence.

SEQ ID NO: 8 is complete mouse CX3C amino acid sequence.

SEQ ID NO: 9 is Gro amino acid sequence.

SEQ ID NO: 10 is Ltn amino acid sequence.

SEQ ID NO: 11 is MIP-10 amino acid sequence.

All references cited herein are incorporated herein by reference to thesame extent as if each individual publication or patent application wasspecifically and individually indicated to be incorporated by referencein its entirety for all purposes.

Many modifications and variations of this invention can be made withoutdeparting from its spirit and scope, as will be apparent to thoseskilled in the art. The specific embodiments described herein areoffered by way of example only, and the invention is to be limited onlyby the terms of the appended claims, along with the full scope ofequivalents to which such claims are entitled.

1. A method for blocking binding of a CX3C chemokine polypeptide to acell that expresses a receptor for the chemokine; wherein said chemokinepolypeptide a) chemoattracts peripheral blood monocytes or T-cells, b)consists of an amino acid sequence comprising at least 95% amino acidsequence identity to the amino acid sequence set forth in SEQ ID NO: 8,and c) comprises a chemokine domain identical to residues 25 to 100 ofthe amino acid sequence set forth in SEQ ID NO: 8; comprising contactingsaid chemokine polypeptide with an antibody or antigen-binding fragmentthereof that specifically binds to said chemokine polypeptide.
 2. Themethod of claim 1 wherein said antibody or antigen-binding fragmentthereof is detectably labeled.
 3. The method of claim 1 wherein saidantibody or antigen-binding fragment thereof is a polyclonal antibody.4. The method of claim 1 wherein said antibody or antigen-bindingfragment thereof is a monoclonal antibody.
 5. The method of claim 1wherein said antibody or antigen-binding fragment thereof is a Fab. 6.The method of claim 1 wherein said antibody or antigen-binding fragmentthereof is a F(ab)₂.